Biologically active spermidine analogues, pharmaceutical compositions and methods of treatment

ABSTRACT

Polyamines having the formula:  
                 
 
     or a salt thereof with a pharmaceutically acceptable acid wherein: R 1 -R 5  may be the same or different and are alkyl, aryl, aryl alkyl, cycloalkyl or hydrogen; at least one of R 1  and R 2  and at least one of R 4  and R 5  are not hydrogen, and any of the alkyl chains may optionally be interrupted by at least one etheric oxygen atom, excluding N 1 ,N 3 -diethylspermidine and N 1 ,N 3 -dipropylspermidine; and  
     A and B are bridging groups which effectively maintain the distance between the nitrogen atoms such that the polyamine: (i) is capable of uptake by a target cell upon administration of the polyamine to a human or non-human animal; and (ii) upon uptake by the target cell, competitively binds via an electrostatic interaction between the positively charged nitrogen atoms to substantially the same biological counter-anions as the intracellular natural polyamines in the target cell.

[0001] The research which led to the completion and reduction topractice of the present invention was supported in part by Grant No.R01-DK49108 awarded by the National Institutes of Health (NIH). The U.S.Government has certain rights in and to the invention described andclaimed herein.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to novel polyamines useful asactive ingredients in pharmaceutical compositions and therapeuticmethods of treatment.

[0004] 2. Description of the Prior Art

[0005] Because of the sustained increases in polyamine biosynthesis inpre-neoplastic and neoplastic tissues, a great deal of attention hasbeen directed to the polyamine biosynthetic network as a target inanti-neoplastic strategies [Pegg, “Polyamine Metabolism and ItsImportance in Neoplastic Growth and as a Target for Chemotherapy,”Cancer Res., Vol. 48, pages 759-774 (1988); and Marton et al,“Directions for Polyamine Research,” J. Cell Biochem., Vol. 45, pages7-8 (1991)]. Initial work focused on the design and synthesis ofcompounds which would inhibit L-ornithine decarboxylase (ODC) [Bey etal, “Inhibition of Basic Amino Acid Decarboxylases Involved in PolyamineBiosynthesis,” Inhibition of Metabolism Biological Significance andBasis for New Therapies, McCann et al, eds.; Academic Press: Orlando,Fla., pages 1-32 (1987)] and S-adenosyl-L-methionine decarboxylase(AdoMetDC) [Pegg, Cancer Res., Vol. 48, supra; and Williams-Ashman etal, “Methylglyoxal Bis(guanylhydrazone) as a Potent Inhibitor ofMammalian and Yeast S-Adenosylmethionine Decarboxylases,” Biochem.Biophys. Res. Commun., Vol. 46, pages 288-295 (1972)]. Some success wasachieved through this approach in that difluoromethylornithine (DFMO),an ODC inhibitor, and methylglyoxyl-bis(guanylhydrazone) (MGBG), anAdoMetDC inhibitor, were effective against both in vivo and in vitrotumors [Sunkara et al, “Inhibitors of Polyamine Biosynthesis: Cellularand In Vivo Effects on Tumor Proliferation,” Inhibition of PolyamineMetabolism Biological Significant Cause and Basis for New Therapies,McCann et al, eds.; Academic Press: Orlando, Fla., pages 121-140 (1987);and Pegg et al, “S-Adenosylmethionine Decarboxylase as an Enzyme Targetfor Therapy,” Pharmacol. Ther., Vol. 56, pages 359-377 (1992)]. However,clinical trials did not mirror the success realized in the modelsystems; the drug either was too toxic as with MGBG [Pegg et al,Biochem. Pharmacol., Vol. 27, pages 1625-1629 (1978)] or was unable toshow significant impact on tumors in humans as with DFMO [Schecter etal, “Clinical Aspects of Inhibition of Ornithine Decarboxylase withEmphasis on the Therapeutic Trials of Eflornithine (DFMO) in Cancer andProtozoan Diseases,” Inhibition of Polyamine Metabolism. BiologicalSignificance and Basis for New Therapies, McCann et al, eds.; AcademicPress: Orlando, FL, pages 345-364 (1987)]. One of the problems with thetarget enzymes ODC and AdoMetDC is associated [Seiler et al, “PolyamineTransport in Mammalian Cells,” Int. J. Biochem., Vol. 22, pages 211-218(1990)] with their very short half-lives, i.e., about 20 minutes. Thiscan translate into a protracted exposure requirement for patients whichis a less than desirable situation. Nonetheless, both DFMO and MGBGserved well as proof of principle that the polyamine biosyntheticnetwork was an excellent target in the design of anti-cancer drugs.

[0006] It would thus be desirable to design polyamine analogues whichwould be incorporated via the polyamine transport apparatus and, once inthe cell, would find their way to the same subcellular distributionsites as the normal polyamines do, but would be unable to be furtherprocessed [Jännë et al, “Polyamines in Rapid Growth and Cancer,”Biochim. Biophys. Acta, Vol. 473, page 241 (1978); and Porter et al,“Enzyme Regulation as an Approach to Interference with PolyamineBiosynthesis—an Alternative to Enzyme Inhibition,” Enzyme Regul., Vol.27, pages 57-79 (1988)]. They would appear enough like the naturalpolyamines to shut down polyamine enzymes just as when the cells areexposed to exogenous spermine.

[0007] Thus, a series of terminally N-alkylated tetraamines, whichexhibit anti-neoplastic activity against a number of murine and humantumor lines both in vitro and in vivo, were assembled [Bergeron et al,“Synthetic polyamine analogues as antineoplastics,” J. Med. Chem., Vol.31, pages 1183-1190 (1988); Bergeron et al, “AntiproliferativeProperties of Polyamine Analogues: a Structure-Activity Study,” J. Med.Chem., Vol. 37, pages 3464-3476 (1994); Bernacki et al, “AntitumorActivity of N,N′-Bis(ethyl)spermine Homologues Against Human MALME-3Melanoma Xenografts,” Cancer Res., Vol. 52, pages 2424-2430 (1992);Porter et al, “Biological Properties of N⁴-Spermidine Derivatives andTheir Potential in Anti-cancer Chemotherapy,” Cancer Res., Vol. 42,pages 4072-4078 (1982); and Porter et al, “Biological Properties of N⁴-and N¹,N⁸-Spermidine Derivatives in Cultured L1210 Leukemia Cells,”Cancer Res., Vol. 45, pages 2050-2057 (1985)]. These tetraamines havebeen shown to utilize the polyamine transport apparatus forincorporation [Bergeron et al, J. Med. Chem., Vol. 37, supra; and Porteret al, “Aliphatic Chain Length Specific of the Polyamine TransportSystem in Ascites L1210 Leukemia Cells,” Cancer Res., Vol. 44, pages126-128 (1984)], deplete polyamine pools [Bergeron et al, “Role of theMethylene Backbone in the Antiproliferative Activity of PolyamineAnalogues on L1210 Cells,” Cancer Res., Vol. 49, pages 2959-2964(1989)], drastically reduce the level of ODC [Pegg et al, “Control ofOrnithine Decarboxylase Activity in α-Difluoromethylornithine-ResistantL1210 Cells by Polyamines and Synthetic Analogues,” J. Biol. Chem., Vol.263, pages 11008-11014 (1988); and Porter et al, “Relative Abilities ofBis(ethyl) Derivatives of Putrescine, Spermidine and Spermine toRegulate Polyamines Biosynthesis and Inhibit L1210 Leukemia CellGrowth,” Cancer Res., Vol. 47, pages 2821-2825 (1987)] and AdoMetDCactivities [Pegg et al, J. Biol. Chem., Vol. 263, supra; and Porter etal, Cancer Res., Vol. 47, supra] and in some cases to up-regulatespermidine/spermine/N¹-acetyltransferase (SSAT) [Pegg et al, “Effect ofN¹,N¹²-Bis(ethyl)spermine and Related Compounds on Growth and PolyamineAcetylation, Content and Excretion in Human Colon Tumor Cell,” J. Biol.Chem., Vol. 264, pages 11744-11749 (1989); Casero et al, “DifferentialInduction of Spermidine/Spermine N¹-Acetyltransferase in Human LungCancer Cells by the Bis(ethyl)polyamine Analogues,” Cancer Res., Vol.49, pages 3829-3833 (1989); Libby et al, “Major Increases inSpermidine/Spermine-N¹-Acetyltransferase by Spermine Analogues and TheirRelationship to Polyamine Depletion and Growth Inhibition in L1210Cells,” Cancer Res., Vol. 49, pages 6226-6231 (1989); Libby et al,“Structure-Function Correlations of Polyamine Analog-Induced Increasesin Spermidine/Spermine Acetyltransferases Activity,” Biochem.Pharmacol., Vol. 38, pages 1435-1442 (1989); Porter et al, “CorrelationsBetween Polyamine Analog-Induced Increases in Spermidine/SpermineN-Acetyltransferase Activity, Polyamine Pool Depletion and GrowthInhibition in Human Melanoma Cell Lines,” Cancer Res., Vol. 51, pages3715-3720 (1991); Fogel-Petrovic et al, “Polyamine and Polyamine AnalogRegulation of Spermidine/Spermine N¹-Acetyltransferase in MALME-3M HumanMelanoma Cells,” J. Biol. Chem., Vol. 268, pages 19118-19125 (1993); andShappell et al, “Regulation of Spermidine/Spermine N¹-Acetyltransferaseby Intracellular Polyamine Pools-Evidence for a Functional Role inPolyamine Homeostasis,” FEBS Lett., Vol. 321, pages 179-183 (1993)].Interestingly, on incorporation of the tetraamine analogues, the totalpicoequivalents of charge associated with the analogues, as well as thenatural polyamines, is maintained for about 24 hours. Thus, as the cellis incorporating n picoequivalents of drug, it is excreting npicoequivalents of natural polyamines.

[0008] Very small structural alterations in these spermine analogues andhomologues result in substantial differences in their biologicalactivity [Bergeron et al, Cancer Res., Vol. 49, supra]. For example,while the tetraamines N¹,N¹²-diethylspermine (DESPM),N¹,N¹¹-diethylnorspermine (DENSPM) and N¹,N¹⁴-diethylhomospermine(DEHSPM) suppress ODC and AdoMetDC to about the same level at equimolarconcentrations, the effect of both DESPM and DEHSPM on cell growthoccurs earlier than that observed for DENSPM. The K_(i) value of DENSPMis over 10 times as great [Bergeron et al, Cancer Res., Vol. 49, supra]as those of DESPM and DEHSPM for the polyamine transport system.However, the most notable difference between the three analogues isrelated to their ability to stimulate SSAT [Casero et al, Cancer Res.,Vol. 49, supra; Libby et al, Cancer Res., Vol. 49, supra; Libby et al,Biochem. Pharmacol., Vol. 38, supra; and Porter et al, Cancer Res., Vol.51, supra]. The tetraamine DENSPM up-regulates SSAT by 1200 fold inMALME-3 cells, while DESPM and DEHSPM stimulate SSAT by 250- and30-fold, respectively [Porter et al, Cancer Res., Vol. 51, supra]. Thus,the impact of the tetraamine compounds on cell growth was shown to bedependent on: the distance between the nitrogens; the nature of theterminal alkyl substituents [Bergeron et al, J. Med. Chem., Vol. 37,supra] and, most importantly, on the charge status of the molecules[Bergeron et al, “The Role of Charge in Polyamine Analogue Recognition,”J. Med. Chem., Vol. 38, pages 2278-2285 (1995)].

[0009] It was decided to establish whether or not a similar structureactivity relationship exists for triamines, i.e., analogues ofspermidine. The importance of this issue is underscored by thetremendous difference in toxicity between the triamines and tetraaminesin general. Triamines are much less toxic, thus making them ofpotentially useful therapeutic value [Bergeron et al, “Metabolism andPharmacokinetics of N¹,N¹¹-Diethylnorspermine,” Drug Metab. Dispos.,Vol. 23, pages 1117-1125 (1995)].

[0010] It is, therefore, an object of the present invention to providecertain novel triamines possessing biological activity, in particular,anti-neoplastic activity.

SUMMARY OF THE INVENTION

[0011] This and other objects are realized by the present invention, oneembodiment of which relates to polyamines not occurring in nature havingthe formula:

[0012] or a salt thereof with a pharmaceutically acceptable acidwherein:

[0013] R₁-R₅ may be the same or different and are alkyl, aryl, arylalkyl, cycloalkyl or hydrogen; at least one of R₁ and R₂ and at leastone of R₄ and R₅ are not hydrogen, and any of the alkyl chains mayoptionally be interrupted by at least one etheric oxygen atom, excludingN¹,N³-diethylspermidine and N¹,N³-dipropylspermidine; and

[0014] A and B may be the same or different and are bridging groupsincluding unsubstituted heterocyclic bridging groups which effectivelymaintain the distance between the nitrogen atoms such that thepolyamine: (i) is capable of up-take by a target cell uponadministration of the polyamine to a human or non-human animal; and (ii)upon uptake by the target cell, competitively binds via an electrostaticinteraction between the positively charged nitrogen atoms tosubstantially the same biological counter-anions as the intracellularnatural polyamines in the target cell, provided that where A or B is aheterocyclic bridging group, the bridging group is an unsubstitutedheterocyclic group incorporating said N¹, N² or N³ atoms in theheterocyclic ring as an unsubstituted N atom; the polyamine, uponbinding to the biological counter-anion in the cell, functions in amanner biologically different than the intracellular polyamines.

[0015] A further embodiment of the invention concerns a pharmaceuticalcomposition in unit dosage form comprising a pharmaceutically acceptablecarrier and a pharmaceutically effective amount of a polyamine asdescribed above or a salt thereof with a pharmaceutically acceptableacid.

[0016] An additional embodiment of the invention comprises a method oftreating a human or non-human patient in need thereof comprisingadministering thereto a pharmaceutically effective amount of a polyaminedescribed above or a salt thereof with a pharmaceutically acceptableacid.

[0017] Other embodiments of the invention will become apparent from thefollowing detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIGS. 1-5 depict reaction schemes for the syntheses of thepolyamines of the invention.

[0019] FIGS. 6(a) and 6(b) depict the structure-activity relationshipbetween triamine analogues and tetraamine analogues, respectively, andSSAT up-regulation.

[0020]FIG. 7 elaborates the metabolic transformation of the triamineanalogues.

[0021]FIG. 8 depicts the structure-activity relationship between thetriamine analogues and K_(i) values.

[0022]FIG. 9(a) represents the structure-activity relationship betweenthe triamine analogues and the IC₅₀ values.

[0023]FIG. 9(b) illustrates the structure-activity relationship betweenthe tetraamine analogues and the IC₅₀ values.

DETAILED DESCRIPTION OF THE INVENTION

[0024] In the polyamines of the invention, as described in the abovestructural formula, R₁-R₆ may be alkyl, e.g., methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl; aryl, e.g., phenyl,p-tolyl, 2,4,6-trimethylphenyl; aryl alkyl, e.g., benzyl, α-phenethyl,β-phenethyl; cycloalkyl, e.g., cyclohexyl, cyclobutyl, cyclopentyl,cycloheptyl; any of the foregoing wherein the alkyl chain is interruptedby etheric oxygen, e.g., CH₃O(CH₂)₂—, CH₃O(CH₂)₂O(CH₂)₂—, CH₃O(CH₂)₂O(CH₂)₂O(CH₂)₂—; or hydrogen.

[0025] Except where R₁-R₆ are hydrogen or etheric substituents, each arehydrocarbyl and may have from about 1 to about 10 carbon atoms, it beingunderstood that the size of the substituents will be tailored in eachcase to ensure that the polyamine is capable of uptake by the targetcell and, upon uptake, will competitively bind with the intracellularcounter-anions as described above.

[0026] The bridging groups A and B may be the same or different and maybe alkylene having 1-8 carbon atoms, e.g., methylene, trimethylene,tetramethylene, pentamethylene; branched alkylene, e.g.,—CH(CH₃)CH₂CH₂—, —CH₂CH(CH₃)CH₂—, —CH(CH₃)CH₂CH₂—, —CH₂CH(CH₃)CH₂CH₂—;arylalkylene, e.g., —CH(Ph)CH₂CH₂—, —CH₂CH(Ph)CH₂—, —CH(Ph)CH₂CH₂CH₂—,—CH₂CH(Ph)CH₂—CH₂—; cycloalkylene, e.g., cyclohexylene, cis- andtrans-1,3-cyclohexylene, 1,4-cyclohexylene, 1,3-cyclopentylene;heterocyclic groups which incorporate within the ring one of thenitrogen atoms of the polyamine [e.g.,

[0027] it being understood that the heterocyclic nitrogen group may belocated at the terminal end(s) or within the interior of the polyamine.

[0028] Those skilled in the art will appreciate that it is onlynecessary that the bridging groups be selected so as to ensure uptake bythe cell and competitive binding to the intracellular counter-anion asdescribed above.

[0029] At physiological pH's, the naturally occurring polyamines and theanalogs of the present invention are largely in a protonated state[Bergeron et al, “Hexahydropyrimidines as masked spermidine vectors indrug delivery,” Bioorg. Chem., Vol. 14, pages 345-355 (1986)]. At acellular level, these polycations can bind to a collection of singleunconnected anions or to anions tethered to a single biomolecule, e.g.,the phosphates on a nucleic acid.

[0030] If there is any significance to the role of charge interaction inthe biological properties of the polyamine analogs, alterations in thepolyamine methylene backbone should have significant impact on thecompound's biological properties. In fact, the significance of chargeand the length of the methylene bridges separating the cations in thebiological properties of the polyamine analogs has been demonstrated.

[0031] Among the most preferred polyamines of the invention are those ofthe following formula:

R₁—N¹H—(CH₂)_(m)—N²H—(CH₂)—N³H—R₂

[0032] wherein: R₁ and R₂ may be the same or different and are H oralkyl; preferably having, at most, 10 carbon atoms; most preferably,methyl, ethyl and n-propyl (with the proviso that both R₁ and R₂ may notbe H); and m and n may be the same or different and are 3, 4 or 5.

[0033] Exemplary of preferred polyamines of the invention are:

[0034] dimethylnorspermidine (DMNSPD)

[0035] monoethylnorspermidine (MENSPD)

[0036] diethylnorspermidine (DENSPD)

[0037] monopropylnorspermidine (MPNSPD)

[0038] dipropylnorspermidine (DPNSPD)

[0039] dimethylspermidine (DMSPD)

[0040] monoethylspermidine [(MESPD)N1]

[0041] monoethylspermidine [(MESPD)N8]

[0042] diethylspermidine (DESPD)

[0043] monopropylspermidine (MPSPD)N1]

[0044] monopropylspermidine [(MPSPD)N8]

[0045] dipropylspermidine (DPSPD)

[0046] dimethylhomospermidine (DMHSPD)

[0047] diethylhomospermidine (DEHSPD)

[0048] monopropylhomospermidine (MPHSPD)

[0049] dipropylhomospermidine (DPHSPD)

[0050] CH₃NH(CH₂)₄NH(CH₂)₅NHCH₃ [DM(4,5)]

[0051] CH₃CH₂NH(CH₂)₄NH(CH₂)₅NHCH₂CH₃ [DE (4, 5)]

[0052] CH₃(CH₂)₂NH(CH₂)₄NH(CH₂)₅NH(CH₂)₂CH₃ [DP(4, 5)]

[0053] CH₃NH(CH₂)₅NH(CH₂)₅NHCH₃ [DM(5,5)]

[0054] CH₃CH₂NH(CH₂)₅NH(CH₂)₅NHCH₂CH₃ [DE(5,5)]

[0055] CH₃(CH₂)₂NH(CH₂)₅NH(CH₂)₅NH(CH₂)₂CH₃ [DP(5,5)]

[0056] It will be understood by those skilled in the art that thepolyamines of the present invention may be employed to effect anydesired biological effect mediated by the polyamine biosynthetic networkor system, e.g., anti-neoplastic, anti-viral, anti-psoriasis,anti-inflammatory, anti-arrhythmic, etc.

[0057] For the purposes of a detailed description of a preferredembodiment of the invention, however, the activity of a representativenumber of polyamines against tumor cells sensitive thereto will bedescribed.

[0058] The triamines of the invention described hereinbelow can beenvisioned as belonging to one of two families of polyamines having thestructural formula:

[0059] One family of polyamines can be characterized as havingsymmetrical methylene backbones, i.e., wherein m=n.

[0060] The other family is unsymmetrical, i.e., m≠n.

[0061] Synthesis of Triamines.

[0062] The two families of triamines were synthesized: (1) those withsymmetrical methylene backbones, i.e., derived from the parentpolyamines norspermidine (3,3), homospermidine (4,4) or the longertriamine (5,5) [wherein (3,3), (4,4) and (5,5) refer to the number ofmethylene groups, i.e., (m,n)], with an alkyl group at one or bothterminal nitrogens; and (2) those with unsymmetrical methylenebackbones, i.e., from the parent polyamines spermidine (3,4) or the(4,5) triamine, with an alkyl group at one or both terminal nitrogens(Table 1). The numbers in parentheses refer to the number of methylenesseparating successive nitrogens. In the case of theN^(α),N^(ω)-disubstituted norspermidine (m=3, n=3) and spermidine (m=3,n=4) analogues, the commercially available triamines norspermidine(NSPD) (1) and spermidine (SPD) (7) were reacted with mesitylenesulfonylchloride (3 equiv) under biphasic conditions (CH₂Cl₂/dilute NaOH) togive 30 [Bergeron et al, Drug Metab. Dispos., Vol. 23, supra], and 31,respectively (step e) (FIG. 1, Scheme 1). These trisulfonamides weredeprotonated with NaH in DMF and treated with an excess of theappropriate primary alkyl iodide to make intermediates 43, 44 and 46-49(step f). Finally, the mesitylenesulfonyl blocking groups were cleanlyremoved under reductive conditions utilizing 30% HBr in HOAc and phenolin CH₂Cl₂ (step g) to give terminal dimethyl-(2, 8), diethyl-(4, 11),and dipropyl-(6, 14) NSPD and SPD, respectively, which were isolated astheir recrystallized trihydrochloride salts.

[0063] The symmetrical triamines homospermidine (HSPD) (15) (4,4) and1,7,13-triazatridecane (24) (5,5), which were not commerciallyavailable, and their terminally dialkylated derivatives were synthesizedby a segmented synthesis (FIG. 1, Scheme 1). Mesitylenesulfonamide (35)[Bergeron et al, Drug Metab. Dispos., Vol. 23, supra] was dialkylatedwith either N-(4-bromobutyl)phthalimide to give 37 (step i) or with5-chlorovaleronitrile to furnish 39 (step c). Hydrogenation of the cyanogroups of 39 with Raney nickel in methanolic ammonia gaveN,N-bis(5-aminopentyl)mesitylenesulfonamide (42) (step d), whichprovided 5,5-triamine 24 in good yield by treatment with 30% HBr in HOAc(step g). Use of the aromatic imide blocking group in 37 avoided thesolubility problems during attempted hydrogenation (Raney nickel,methanolic NH₃) of N,N-bis(3-cyanopropyl)mesitylenesulfonamide.Hydrazinolysis of 37 in refluxing EtOH (step j) led toN,N-bis(4-aminobutyl)mesitylenesulfonamide (40). HSPD (15) itselfresulted from reductive deprotection of monosulfonamide 40 (step g).Terminal diamines 40 and 42 were converted to theirmesitylenesulfonamides 32 and 34, respectively, (step e) and werealkylated with the appropriate primary halide (step f). Hydrogenbromide-promoted deprotection of masked analogues 50, 52 and 55-57yielded DMHSPD (16), DPHSPD (19), DM(5,5) (25), DE(5,5) (26) and DP(5,5)(27), respectively.

[0064] N¹,N⁹-Diethylhomospermidine (DEHSPD) (17) was made by aconvergent route (FIG. 1, Scheme 1). Alkylation of sulfonamide 35 withN—(4-bromobutyl)-N-ethylmesitylenesulfonamide (58) [Bergeron et al, J.Med. Chem., Vol. 37, supra] (2 equiv) led to triprotected analogue 51(step h), which was unmasked with HBr/HOAc, giving DEHSPD (17) (step g).3,8,14-Triazahexadecane [DE(4,5)] (22), the terminally diethylatedanalogue of the unsymmetrical 4,5-triamine, was assembled fromN-(tert-butoxycarbonyl)-N-mesitylenesulfonamide (59), a diprotectedammonia synthon [Bergeron et al, J. Med. Chem., Vol. 37, supra] (FIG. 2,Scheme 2). Alkylation of reagent 59 withN-(4-bromobutyl)-N-ethylsulfonamide (58) (NaH/DMF) (step a) gavetriprotected monoethylputrescine 62. The BOC group of 62 was removedwith trifluoroacetic acid (TFA) (step c). The resulting sulfonamide 63was alkylated with N-(5-bromopentyl)-N-ethylmesitylenesulfonamide (61)(step a), which was made from ethylsulfonamide 60 and excess1,5-dibromopentane (NaH/DMF) (step b), to generate fully protectedtriamine 64. Deprotection of the amino groups of 64 with HBr led to thediethylated analogue 22 (step d).

[0065] The 4,5-triamine 1,6,12-triazadodecane (20) and its dialkylatedanalogues 2,7,13-triazatetradecane [DM(4,5)] (21) and4,9,15-triazaoctadecane [DP(4,5)] (23) were produced by a segmentedsynthesis (FIG. 1, Scheme 1). Consecutive monoalkylation of sulfonamide35 with 4-bromobutyronitrile (step a) and 5-bromovaleronitrile (step b)generated dinitrile 38. The cyano groups of 38 were reduced in a Parrshaker with Raney nickel in methanolic ammonia (step d), resulting inprimary amine 41. Cleavage of the sulfonyl group of 41 with HBr (step g)produced the parent 4,5-triamine 20. Treatment of 41 withmesitylenesulfonyl chloride (2 equiv) gave 33 (step e), which wasterminally dialkylated with iodomethane to 53 or with 1-iodopropane to54 (step f). Unmasking the amino groups led to dimethylated anddipropylated 4,5-analogues 21 and 23, respectively (step g).

[0066] N¹-Propylnorspermidine (MPNSPD) (5) was made by treatingtrimesitylenesulfonyl NSPD 30 [Bergeron et al, Drug Metab. Dispos., Vol.23, supra] with 1-iodopropane (1 equiv/NaH/DMF), and isolating 45 fromthe statistical mixture of mono- and di-alkylated products by flashcolumn chromatography (step f) (FIG. 1, Scheme 1). Since SPD isunsymmetrical, reaction of its trisulfonamide 31 with a primary alkyliodide (1 equiv) would lead to N¹- and N⁸-monoalkylated products, whichmay be difficult to separate. Thus, the synthesis of both SPD and theHSPD monopropyl analogues required a fragment synthesis (FIG. 3, Scheme3). N-Propylmesitylenesulfonamide (65) was converted to 3-bromopropyl 66or 4-bromobutyl reagent 67, with the required dibromoalkane in excess(NaH/DMF). Triphenylmethyl chloride was stirred at room temperature witheither 1,3-diaminopropane or 1,4-diaminobutane (5 equiv) in CH₂Cl₂ (stepc), resulting in N¹-tritylated-trimethylenediamine 68 or -putrescine 69.Sulfonation of 68 and 69 occurred at the primary nitrogen and not nextto the bulky triphenylmethyl group to give N,N′-disubstituted diamines70 and 71, respectively (step a).

[0067] Reaction of the anions of 70 or 71 with the appropriate bromide66 or 67 resulted in regiospecific N-alkylation at the sulfonamideterminus. Specifically, reaction of 70 with 67 gave 73, and 71 plus 66or 67 led to 72 or 74, respectively.

[0068] The protecting groups of 45 and 72-74 were removed simultaneouslywith Hbr in HOAc/PhOH, resulting in MPNSPD (5), MPSPD(N¹) (12),MPSPD(N⁸) (13) and MPHSPD (18), respectively.

[0069] Both N¹- (9) and N⁸-ethylspermidine (10) were obtained fromreduction of the requisite monoacetylspermidine with lithium aluminumhydride in hot THF, thus completing the synthesis of the triamineseries.

[0070] Tetraamine analogue N¹,N¹¹-dipropylnorspermine (DPNSPM) (28) wasaccessed from commercially available norspermine (FIG. 4, Scheme 4).Bis-alkylation of the tetrasulfonamide dianion of 75 [Bergeron et al, J.Med. Chem., Vol. 37, supra] with 1-iodopropane (step a) and facileremoval of the mesitylenesulfonyl blocking groups of 76 with HBr (stepb) generated DPNSPM (28).

[0071] The longer polyamine 3,9,14,20-tetraazadocosane [DE(5,4,5)] (29),the terminally diethylated derivative of the unknown (5,4,5) tetraamine,was synthesized in three high yield steps by the segmenting method (FIG.5, Scheme 5). N-Ethylmesitylenesulfonamide (60) [Schreinemakers, Recl.Trav. Chim. Pays-Bas Belg., Vol. 16, pages 411-424 (1897)] wasdeprotonated (NaH/DMF) and treated with 1,5-dichloropentane (10 equiv),resulting in alkyl chloride 77 (step a).N¹,N⁴-Bis(mesitylenesulfonyl)putrescine (78) [Bergeron et al, J. Med.Chem., Vol. 37, supra] was alkylated with synthon 77 to give maskedtetraamine 79 (step b). The four blocking groups were removed with HBr(step c) to furnish DE(5,4,5) 29 as its crystalline tetrahydrochloridesalt. Biological Evaluations. In summarizing the biological propertiesof the polyamine analogues, the results are separated into three sets ofmeasurements: the 48- and 96-hour IC₅₀ values against L1210 cells andthe corresponding K_(i) values for the polyamine transport apparatus(Table 1); the effect on polyamine pools (Table 2); and the impact onODC, AdoMetDC and SSAT (Table 3). The compounds are arranged in sets byincreasing length, e.g., norspermidine, spermidine, homospermidine,(4,5)- and (5,5)-triamines. Each set is ordered in terms of the size ofthe terminal alkyl groups. While the IC₅₀ and K_(i) values of DESPD andits impact on polyamine pools, ODC, AdoMetDC and SSAT have beenpreviously reported [Porter et al, Cancer Res., Vol. 45, supra], themeasurements on this compound were repeated so that the appropriatepositive control and not a historical control would be in place. Inorder to showcase the importance of the polyamine's overall chain lengthin structure-activity relationships, there is included a briefcommentary of results of tetraamine analogues [Bergeron et al, J. Med.Chem., Vol. 37, supra] where available. Thus, numbers included inparentheses in the tables represent the values for the correspondingtetraamine analogues. A brief discussion is also presented on themetabolic profile of the triamines and on the cationic conservation ofcharge the cell maintains as defined by the polyamines. Finally, acomparison of the acute and chronic in vivo toxicities of several keytriamines and tetraamines is presented.

[0072] Antiproliferative Activity—IC₅₀ of L1210 cells.

[0073] As shown in Table 1, NSPD is the most active among the NSPDfamily of analogues with an IC₅₀ of 0.9 μM at 48 hours and 0.5 μM at 96hours. This activity is probably related to the fact that this triaminecan easily be converted to toxic metabolites [Alarcon et al, “Evidencefor the Formation of Cytotoxic Aldehyde Acrolein from EnzymaticallyOxidized Spermine or Spermidine,” Arch. Biochem. Biophys., Vol. 137,pages 365-372 (1970)]. All of the alkylated norspermidine analogues haveIC₅₀ values >100 μM at 48 hours. At 96 hours, the IC₅₀ values range from3.5 to >100 μM with an order of DMNSPD<DENSPD<DPNSPD<MENSPD and MPNSPD(most to least active). Thus, in this family, terminal dialkylation withsmaller groups increases the compound's activity, while triamines with asingle alkyl group are less active than the corresponding compound withbis N^(α),N^(ω)-alkyl substitution. In contrast, analogues of thetetraamine norspermine, although also inactive at 48 hours, were moreactive than the corresponding triamines at 96 hours. Moreover, whethernorspermine was symmetrically substituted with methyl or ethyl groups orhad a single ethyl fixed to one of the terminal nitrogens wasinsignificant relative to the 96-hour IC₅₀ values, which were around 2μM.

[0074] At 48 hours, SPD and all of its analogues had an IC₅₀ of at least100 μM. Unlike NSPD, and not surprisingly, SPD is the least activecompound in its family with IC₅₀ values above 100 μM at both 48 and 96hours. At 96 hours, DMSPD and DESPD are substantially more active thanDPSPD. When an ethyl group was removed from either end of DESPD, amonoalkylated analogue was produced with lower activity than DESPD, byone to two orders of magnitude. It is interesting that monoalkylation ofSPD by ethyl or propyl at different ends result in very differentactivities. At 96 hours, with an IC₅₀ of 4 μM, MESPD(N¹) was about 10times more active than MESPD(N⁸). The same trend was found, although toa lesser degree, among the two monopropyl SPD analogues in thatMPSPD(N¹) was more than twice as active as MPSPD(N⁸). Thus, alkylationat the N¹ position results in a higher activity than alkylation at N⁸(Table 1). The spermine analogues had a significant effect on cellgrowth even at 48 hours and at 96 hours, the IC₅₀ concentrations oftetraamines ranged from 0.2 to 0.8 μM, with DESPM<DPSPM<MESPM<DMSPM.Again, in every instance, the tetraamines were more active. It isinteresting that 3.7% of intracellular N¹-MESPD and 6.1% ofintracellular N¹-MPSPD are metabolically converted to the correspondingtetraamines, ME-[3,4,3] and MP-[3,4,3] respectively (Table 4). Given thepotent antiproliferative activity of the tetraamines in general, thismay help explain the enhanced activity of the N¹-alkylspermidines incomparison to the N⁸-alkylspermidines since the latter are notmetabolically converted to tetraamines.

[0075] Among the HSPD analogues, DEHSPD is active at 48 hours with anIC₅₀ of 25 μM. Other analogues' IC₅₀s are at least 100 μM at 48 hours.At 96 hours, all of the IC₅₀s fall into the range from 0.3˜0.9 μM,except for DPHSPD which has an IC₅₀ of 6 μM. Compared to thenorspermidine and spermidine analogues, the homospermidine analogues asa group are more active. With the tetraamines, the most notabledifferences in activity were between the diethyl and dimethyl compounds(Table 1).

[0076] The (4,5) series are the most effective triamines identified. Asthe triamine chain increases in length from (4,5) to (5,5), the activitydecreases at both 48 hours and 96 hours. Specifically, DM(4,5) andDE(4,5) have IC₅₀ values in the 2-6 μM range, while the DP(4,5) has anIC₅₀ around 100 μM at 48 hours. At 96 hours, the IC₅₀ values of bothseries substantially decrease; even DP(4,5) has an IC₅₀<2 μM. Thenumbers are uniformly higher for the (5,5) triamines even at 96 hours.The corresponding tetraamine analogues DE(4,5,4) and DE(5,4,5) are moreactive at both 48 and 96 hours.

[0077] Competitive Uptake Determinations in L1210 cells.

[0078] The ability of the norspermidines, spermidines, homospermidines,4,5- and 5,5-triamines to compete with radiolabeled SPD for uptake wasevaluated (Table 1). The general trend is that the terminally alkylatedtriamines have higher K_(i) values than the unalkylated triamines andare thus less easily taken up by the cell. In the dialkylated series ofspermidines, homospermidines and 4,5-triamines, K_(i) values increase asthe size of the terminal group increases. This is not completely truewith the norspermidines and the 5,5 triamines. The relationship holdswith methyl and ethyl but not for the propyl of the latter two systems.Finally, the number of methylenes separating the amines plays a role indetermining polyamine uptake properties. In general the effectivenesswith which the analogues compete for uptake isspermidines≈homospermidines>4,5 triamines>5,5 triamines>norspermidine.Interestingly, this same trend is observed with the ethylatedtetraamines, spermines≈homospermines>DE(3,4,4)≈DE(4,5,4)>norspermine.TABLE 1 TRIAMINE ANALOGUE STRUCTURES, ABBREVIATIONS, L1210 GROWTHINHIBITION AND TRANSPORT IC₅₀ (μM) Structure Abbreviation 48 hour 96hour K_(i)(μM) Norspermidines 1

NSPD 0.9 0.5 7.2 2

DMNSPD >100 (>100) 3.5-6.0  (2.5) 60  (5.6) 3

MENSPD >100 (>100) >100  (2.5) 34  (7.7) 4

DENSPD >100 (>100) 10  (2) 250 (17) 5

MPNSPD >100 ˜100 33 6

DPNSPD >100 (>100) 60 (18) 125 (11) Spermidine 7

SPD >100 >100 2.2 8

DMSPD >100 (>100) 1.5-1.8  (0.75) 5.1  (1.1) 9

MESPD(N1) >100  (99) 3.0-5.0  (0.33) 8.6  (1.7) 10

MESPD(N8) >100 40 7 11

DESPD ˜100  (30) 0.6-0.8  (0.18) 19.3  (1.6) 12

MPSPD(N1) >100 20-35 3.0 13

MPSPD(N8) >100 50-60 8.5 14

DPSPD >100   (3) 30-35  (0.2) 25.6  (2.3) Homospermidines 15

HSPD >100 1.7-4.0 3.4 16

DMHSPD >100 (>100) 0.9  (0.32) 5.5  (0.97) 17

DEHSPD 18-25   (0.2) 0.3-0.4  (0.07) 19  (1.4) 18

MPHSPD 100 0.5-0.7 5.0 19

DPHSPD >100 6.0 67 4,5-triamines 20

4,5-Triamine >100 0.15-0.20 1.4 21

DM(4,5) 2.0 0.11-0.12 21 22

DE(4,5) 3.0-6.0   (0.3) 0.19-0.2   (0.035) 64  (6.0) 23

DP(4,5) ˜100 1.0-1.4 75 5,5-triamines 24

5,5-Triamine ˜100 0.3-0.5 13.8 25

DM(5,5) 15 0.4 133 26

DE(5,5) 10-12   (0.4) 0.65-0.7   (0.03) 174 (16) 27

DP(5,5) >100 6.0 87

[0079] Polyamine Pools.

[0080] The following guidelines were adopted for studying the impact ofthe analogues on polyamine pools (Table 2). The measurements were madeafter a 48-hour exposure to the analogue, and two differentconcentrations of analogue were evaluated in each case. For analogueswhose IC₅₀ concentration exceeded 100 μM at 48 hours, the polyaminepools were determined at 100 and 500 μM. For the other analogues, theeffect on polyamine pools was evaluated at the 48-hour IC₅₀concentration and at 5 times this number.

[0081] At 500 μM, the effects of DMNSPD, DENSPD and MPNSPD on polyaminepools were similar (Table 2), i.e., PUT was depleted below detectablelimits and spermidine was reduced to 6-15% of controls, while sperminelevels were diminished to below 50%. DPNSPD was not as effective as theother norspermidine analogues in depletion of polyamine pools, e.g., at500 μM, PUT was only lowered to 68%, SPD to 71% and no effect on SPMlevel. The dipropyl analogue was similar in behavior to the parentnorspermidine. The corresponding norspermines were again more effective.At 100 μM, the effect of DMNSPM and DENSPM on polyamine pools wassimilar, i.e., putrescine was depleted to below detectable limits andspermidine was reduced to around 5% of controls, while spermine levelswere diminished to 27-36%.

[0082] DMSPD and DESPD at 100 μM depleted PUT to below detectablelimits, SPD to 5%, SPM to 58% and 74% of control, respectively. Themonoalkylated SPD analogues MESPD(N¹), MESPD(N⁸) and MPSPD(N¹) gave asimilar pattern of polyamine pool depletion. At 100 μM, putrescine wasdepleted to below detectable levels, spermidine to 25% and spermine to80%, 84% and 90% of control value. MPSPD(N⁸) was slightly less activethan MPSPD(N¹). At 500 μM, DPSPD reduced PUT to below detectable leveland SPD to around 10% of control. Like DPNSPD, DPSPD showed littlesuppression of SPM levels and possibly even some up-regulation at 100μM. Interestingly, at the level of PUT and SPD suppression, MPSPD (N¹)and MPSPD (N⁸) behave very much like their MESPD counterparts. However,the propyl analogues are slightly less effective at sperminesuppression. The parent amine SPD suppresses PUT, but not SPD or SPM.Again, the corresponding spermines are more effective than thetriamines. At 100 or 500 μM DMSPM or MESPM or at 30 and 150 μM DESPM,putrescine was reduced to below detectable limits, spermidine diminishedto under 2% of control and spermine to under 25%. At 3 μM, DPSPM reducedputrescine to below detection and spermidine to 18%, while the sperminelevel remained at 64% of control. At 15 μM DPSPM, spermidine was furtherreduced to 9% and spermine to 43%. Among the homospermidine analogues,the parent triamine, HSPD, was the most active at polyamine suppression.At 100 μM, PUT was depleted to again below detectable levels, SPD to 4%and SPM to 32%. With all of the HSPD analogues, at 500 μM, the level ofputrescine was diminished to below detectable limits and the SPD levelbelow 10% of control. DMHSPD and DEHSPD had little impact on SPM level,while MPHSPD produced a mild decrease. In the case of cells grown in 100μM and 500 μM DPHSPD, the level of SPM seemed to be increased comparedto the controls. As is usual, the homospermines were more effective thanthe corresponding triamine counterparts. At 100 μM, the homospermineanalogue DMHSPM was similar to the corresponding alkyl spermine in itsability to deplete the polyamines. However, DEHSPM was somewhat lesseffective at suppressing spermine pools in comparison to DESPM.

[0083] Similar results were observed with homospermidine homologues, the(4,5) and (5,5) triamines. At 500 μM, the (4,5) and (5,5) parent aminesdepleted both PUT and SPD below detectable level and SPM to 35% and 20%of control, respectively. DM(4,5) at 10 μM and DE(4,5) at 15 μM reducePUT below detectable limits and SPD to 18% of control. However, neitheris very effective at reducing SPM levels. DP(4,5) even at 500 μM, whileit depletes the cell of PUT, only reduces SPD to 31% of control withpossible stimulation of SPM. Finally, DP(5,5) is only marginally active,requiring a 500 μM concentration to even reduce PUT by 50% and SPD by30% and with no impact on SPM. However, the homospermine homologuesDE(4,5,4) and DE(5,4,5), both of which demonstrated low 48-hour IC₅₀values, 0.3 and 0.4 μM, respectively, were similar to the correspondingtriamines at reducing polyamines. TABLE 2 IMPACT OF TRIAMINE ANALOGUESON POLYAMINE POOLS^(a) Compd Conc.(μM) Put Spd Spm Analogue^(b)Norspermidines 1 NSPD 0.9 38 44 113 1.09 4.5 0 12 83 2.14 2 DMNSPD 100(100) 0 (0) 9 (5) 58 (36) 5.00 (2.14) 500 (500) 0 (0) 6 (3) 48 (27) 5.51(1.84) 4 DENSPD 100 (10) 0 (30) 17 (14) 74 (31) 3.67 (1.59) 500 (100) 0(0) 7 (6) 47 (30) 3.77 (2.44) 5 MPNSPD 100 0 29 56 3.07 500 0 15 49 4.786 DPNSPD 100 (100) 70 (61) 76 (35) 96 (77) 0.49 (0.89) 500 (500) 68 (0)71 (19) 102 (56) 1.24 (1.28) Spermidines 7 SPD 100 0 117 118 — 500 0 145108 — 8 DMSPD 100 (100) 0 (0) 5 (0) 58 (21) 4.96 (1.26) 500 (500) 0 (0)0 (0) 54 (24) 4.89 (1.24) 9 MESPD(N1) 100 (100) 0 (0) 25 (1) 80 (21)4.20 (1.24) 500 (500) 0 (0) 14 (1) 53 (19) 4.73 (1.23) 10 MESPD(N8) 1000 26 84 4.41 500 0 15 61 4.96 11 DESPD 100 (30) 0 (0) 5 (0) 74 (12) 4.61(0.40) 500 (150) 0 (0) 0 (0) 55 (14) 4.20 (1.13) 12 MPSPD(N1) 100 0 2590 3.97 500 0 15 72 4.95 13 MPSPD(N8) 100 0 33 103 3.52 500 0 16 95 5.1614 DPSPD 100 (3) 6 (0) 35 (18) 135 (64) 3.26 (1.12) 500 (15) 0 (0) 12(9) 99 (43) 3.69 (1.09) Homospermidines 15 HSPD 100 0 4 32 3.58 500 0 221 4.44 16 DMHSPD 100 (100) 0 (0) 3 (0) 106 (30) 5.51 (1.49) 500 (500) 0(0) 0 (0) 106 (27) 5.85 (1.03) 17 DEHSPD 25 (10) 0 (0) 6 (0) 114 (61)4.61 (2.94) 125 0 3 97 4.69 18 MPHSPD 100 0 2 82 5.16 500 0 0 66 5.86 19DPHSPD 100 0 19 144 3.12 500 0 7 111 3.68 4,5-Triamines 20 4,5 100 0 153 3.02 500 0 0 35 3.20 21 DM(4,5) 2 0 33 112 2.79 10 0 18 111 5.36 22DE(4,5) 3 (0.3) 0 (44) 47 (61) 99 (70) 1.20 (0.26) 15 (1.5) 0 (0) 18 (5)98 (31) 3.40 (0.72) 23 DP(4,5) 100 0 40 119 1.40 500 0 31 121 2.425,5-Triamines 24 5,5 100 0 0 33 2.58 500 0 0 20 2.50 25 DM(5,5) 15 0 33115 2.56 75 0 20 101 3.61 26 DE(5,5) 15 (0.15) 0 (37) 55 (55) 97 (88)1.09 (0.34) 75 (0.75) 0 (0) 23 (10) 73 (58) 1.59 (1.48) 27 DP(5,5) 10059 73 97 0.86 500 51 69 103 1.33

[0084] Impact of Analogues on ODC and AdoMetDC Activities.

[0085] A comparison of the effects of the triamine versus tetraaminepolyamine analogues on ODC and AdoMetDC clearly demonstrates that thetetraamines are more effective at suppressing these enzymes than thecorresponding triamines [Bergeron et al, J. Med. Chem., Vol. 37, supra].Previous studies [Porter et al, “Regulation of Ornithine DecarboxylaseActivity by Spermidine and the Spermine Analogue N¹,N⁸-Bis(ethyl)spermidine,” Biochem. J., Vol. 242, pages 433-440 (1987); and Porter etal, “Combined Regulation of Ornithine and S-AdenosylmethionineDecarboxylase by Spermine and the Spermine AnalogueN¹,N²-Bis(ethyl)-spermine,” Biochem. J., Vol. 268, pages 207-212 (1990)]suggested that the effect of the polyamine analogues on ODC and AdoMetDCis fairly rapid. For example, DESPM induced reduction in ODC activityplateaued at 4 hours and AdoMetDC at 6 hours. On the basis of thesestudies, it was elected to evaluate the impact of the triamines on ODCand AdoMetDC at 4 and 6 hours, respectively.

[0086] The parent triamine norspermidine reduced ODC activity to 11% ofcontrol, the corresponding dimethyl, DMSPD, to 17%, the diethylanalogue, DENSPD, to 80% and the dipropyl compound, DPNSPD, had noeffect on this enzyme (Table 3). Monoethylnorspermidine, MENSPD, wasmore active than the corresponding dialkyl analogue, DENSPD, withreduction to 42 versus 80% of control, as was the monopropyl, MPNSPD,relative to its dipropyl counterpart DPNSPD, with reduction to 33 versus100% of control. In 4 hours, 1 μM DMNSPM, MENSPM or DENSPM reduced ODCactivity to nearly the same extent, to approximately 7% of control. Thetriamines are generally less effective than the correspondingtetraamines at suppressing AdoMetDC, although the differences are not asprofound with the norspermidines versus the norspermines. Thenorspermidines reduce the AdoMetDC to 41-58% of control and thenorspermines to 33-49% of control, except the dipropyls, which are atbest marginally effective.

[0087] The spermidine analogues are less active than the correspondingspermines but more effective at reducing ODC activity than thenorspermidines. At 1 μM DMSPM, MESPM or DESPM, ODC activity was reducedto 10% or less of control, while ODC in DPSPM-treated cells was onlylowered to 52% of controls. The parent triamine spermidine reduces ODCto 16% of control while the alkylated analogues except for DPSPD,diminish ODC activity to between 10-30% of control. Again, the monoalkylanalogues are more effective than the corresponding dialkyl compounds.MESPD(N¹) and MESPD(N⁸) reduce ODC to 10 and 17% versus 30% for DESPD.This property of the monoalkylated analogue is even further accentuatedwith the propylated spermidines MPSPD(N¹) and MPSPD(N⁸) versus DPSPDlowering ODC to 18 and 14% versus 75% of control. Again, when comparingdialkylated compounds, the larger the alkyl substituent the less activethe analogue.

[0088] The spermidine analogues were less effective than thecorresponding norspermidines and spermines at reducing AdoMet activity.The spermidine analogues, except for DPSPD, reduce AdoMetDC to under 70%of control activity. DPSPD has no impact on the enzyme. Again, as withODC, the monoalkylated spermidine compounds were generally more activethan the dialkylated compounds. DMSPM, MESPM or DESPM at 1 μM almostparalleled the ability of the corresponding norspermine analogues tosuppress AdoMetDC with an average reduction to 33% of control, slightlymore active than the spermidines. DPSPM at 1 μM reduced AdoMetDCactivity to 72% of that seen in untreated cells.

[0089] The homospermidines were less active than the correspondinghomospermines at reducing ODC activity, but similar in behavior to thespermidines. Also, consistent with the norspermidine and spermidineresults, the triamines with the larger substituent, propyl, were leasteffective and the monoalkyl compounds were more active than thecorresponding dialkyl ones. Finally, the homospermidine analogues,except for MPHSPD, were not effective at AdoMetDC inhibition andcertainly less active than the corresponding tetraamines.

[0090] Interestingly, adding a methylene unit to DEHSPM to produceDE(4,5,4) resulted in a decrease in ODC suppressing activity. ODC waslowered to only 7% of control with DEHSPM and to 20% of control withDE(4,5,4). This methylene addition had little effect on reduction ofAdoMetDC activity, to about 40% for both. The same phenomenon wasobserved on moving from lower alkylhomospermidines to the dialkyl (4,5)and dialkyl (5,5) compounds, the ODC-suppressing capacity substantiallydecreased while the AdoMetDC properties were similar to those of thehomospermidines. It is noteworthy, however, that the parent(4,5)-triamine demonstrates reasonably effective suppression of ODC andAdoMetDC. The (5,5) parent triamine is an effective and highly selectiveODC antagonist, reducing ODC to 16% of control with little effect onAdoMetDC. Other than this, there is little effect on either ODC orAdoMetDC by (5,5) analogues. The tetraamine analogue DE(5,4,5) is farmore active against both ODC and AdoMetDC than DE(5,5).

[0091] Impact of Triamine Analogues on SSAT Activity.

[0092] While the influence of chain length and terminal substituents aremore monotonic regarding their effect on the analogues' suppression ofODC and AdoMetDC, there are nevertheless some notable structure-activityrelationships for SSAT stimulation. The ability of triamine analogues toup-regulate SSAT in L1210 cells is remarkably sensitive to smallstructural changes (Table 3; FIG. 6a). For example, the diethyltriamines stimulate SSAT 780% for DE(3,3) to a peak of 1380% forDE(3,4), with a decrease to 640% for DE(4,4) and falling to essentiallycontrol values for DE(4,5), 120%, and DE(5,5), 90%. The DE triaminestructure activity curve appears to be shifted to the right from thecorresponding DE tetraamine curve (FIG. 6b). Thus, the DE tetraaminecurve is maximal at 1500% of control for DE(3,3,3) and falls to nearlycontrol value for DE(4,4,4), DE(4,5,4) and DE(5,4,5).

[0093] Substituent changes on the triamines have a profound effect onSSAT stimulation only with the (3,3) and (3,4) compounds. Thedifferences are more compressed for the (4,4) and (4,5) triamines andabsent with (5,5) triamines. In the case of the tetraamines, the (3,3,3)system is the only framework in which a marked effect in SSATstimulation is observed with substituent changes. While there are somechanges with the (3,4,3) backbone, these are again compressed.

[0094] With both the triamines and tetraamines, unlike with ODC andAdoMetDC, there is no relationship between substituent size and SSATup-regulation. However, when clear differences exist between stimulatoryabilities, i.e., (3,3), (3,4), (3,3,3), the ethyl group is clearly thesuperior. TABLE 3 EFFECT OF POLYAMINE HOMOLOGUES ON ORNITHINEDECARBOXYLASE (ODC), S-ADENOSYLMETHIONINE DECARBOXYLASE (AdoMetDC) ANDSPERMIDINE/SPERMINE ACETYLTRANSFERASE (SSAT) IN L1210 CELLS Compd ODCAdoMetDC SSAT Norspermidines 1 NSPD  11  62  150 2 DMNSPD  17 (6)  41(49)  250 (200) 3 MENSPD  42 (5)  58 (33)  390 (410) 4 DENSPD  80 (10) 45 (42)  780 (1500) 5 MPNSPD  33  38  470 6 DPNSPD 100 (79)  99 (70) 220 (460) Spermidines 7 SPD  16  43  160 8 DMSPD  22 (3)  68 (40)  270(300) 9 MESPD(N1)  10 (10)  58 (27)  430 (150) 10 MESPD(N8)  17  54  40011 DESPD  30 (3)  68 (28) 1380 (460) 12 MPSPD(N1)  18  56 1200 13MPSPD(N8)  14  64  500 14 DPSPD  75 (52) 107 (72) 1030 (500)Homospermidines 15 HSPD  11  54  430 16 DMHSPD  20 (4)  86 (45)  510(140) 17 DEHSPD  47 (7)  90 (41)  640 (110) 18 MPHSPD  20  59  570 19DPHSPD  86 123  420 4,5-triamines 20 4,5-triamine  19  57  410 21DM(4,5)  56  71  130 22 DE(4,5) 100 (20)  70 (39)  120 (120) 23 DP(4,5) 83  86  80 5,5-triamines 24 5,5-triamine  16  88  90 25 DM(5,5) 105  97 90 26 DE(5,5) 100 (19) 109 (54)  90 (190) 27 DP(5,5)  73 123  90

[0095] Metabolism.

[0096] In an experiment focused on the impact of DPNSPD (DP-[3,3] inTable 4) on polyamine pools, a substantial unexpected peak appeared inthe chromatogram of treated cells. The suspicious peak was shown tocorrespond to MPNSPD (MP-[3,3] in Table 4) as confirmed by co-elutionwith an authentic sample. The intracellular levels of MPNSPD after a48-hour exposure to DPNSPD was about 50% of intracellular level of theparent compound. Although N-dealkylation had been shown to be animportant step in the metabolism of the alkylated tetraamines DENSPM[Bergeron et al, Drug Metab. Dispos., Vol. 23, supra] and DEHSPM[Bergeron et al, “Metabolism and Pharmacokinetics ofN¹,N¹⁴-Diethylhomospermine,” Drug Metab. Dispos., Vol. 24, pages 334-343(1996)] in vivo in rodents, dogs and man, previous in vitro studies withDEHSPM or DESPM [Bergeron et al, J. Med. Chem., Vol. 37, supra] in L1210cells revealed either little or no N-dealkylation under the conditionsof the experiments. The observation of N-depropylation of DPNSPDcompelled a closer look at the metabolism of the polyamine analogues inL1210 cells. In particular, the significance of the nature of theN-alkyl groups on N-dealkylation was evaluated, in addition to thelength and symmetry of the polyamine backbone. TABLE 4 METABOLICTRANSFORMATION OF POLYAMINE ANALOGUES BY L1210 CELLS Analogue#N-Monodealkylation^ Deaminopropylation^ Elaboration^ DM-[3,3] (100 μM)5000 (100%) no N-demethylation MM-[3,3] (100 μM) 2633 (82.9%) noN-demethylation MM-[3,3] 523 (16.5%) MM-[3]** 20 (0.6%) DE-[3,3,3] (500μM) 2440(95.3%) ME-[3,3,3] 194 (4.7%) DE-[3,3] (500 μM) 3761 (90.7%)ME-[3,3] 194 (4.7%) ME-[3] 192 (4.6%) ME-[3,3] (100 μM) 3051 (78.2%)[3,3]  20 (0.5%) ME-[3] 831 (21.3%) no elaboration of N-Monoalkyl [3,3]DP-[3,3,3] (100 μM)  893 (78.2%) MP-[3,3,3]* 160 (14.0%) MP-[3,3]  89(7.8%) DP-[3,3] (100 μM)  404 (57.7%) MP-[3,3] 214 (30.6%) [3,3]***  64(9.1%) MP-[3]*  18 (2.6%) MP-[3,3] (100 μM) 3019 (94.3%) [3,3] 144(4.5%) MP-[3]  37 (1.2%) no elaboration of N-Monoalkyl [3,3] DM-[3,4](100 μM) 5000 (100%) no N-demethylation DE[3,4] (100 μM) 4041 (96.3%)N8-ME-[4,3]  32 (0.8%) N1-ME-[3,4] 101 (2.4%) N1-ME-[3,4] (100 μM) 3946(96.3%) [3,4] not determined ME-[3,4,3] 150 (3.7%) N8-ME-[4,3] (100 μM)4825 (99.4%) [3,4] not determined ME-[4]  29 (0.6%) no elaboration ofN8-Alkyl [4,3] DP-[3,4,3] (15 μM) 1568 (79.0%) MP-[3,4,3]* 361 (18.2%)N1-MP-[3,4]  57 (2.9%) DP-[3,4] (100 μM) 3260 (90.1%) N8-MP-[4,3] 192(5.3%) N1-MP-[3,4] 166 (4.6%) N1-MP-[3,4] (100 μM) 4238 (93.9%) [3,4]not determined MP-[3,4,3] 275 (6.1%) N8-MP-[4,3] (100 μM) 3549 (99.3%)[3,4] not determined MP-[4]*  26 (0.7%) no elaboration of N8-Alkyl [4,3]DM-[4,4] (100 μM) 5500 (100%) no N-demethylation no exposed aminopropylterminal segment DE-[4,4,4] (100 μM) 4215 (100%) no N-deethylation ″DE-[4,4] (100 μM) 4215 (100%) no N-deethylation ″ DP-[4,4] (500 μM) 3149(87.7%) MP-[4,4] 441 (12.3%) ″ elaboration of N-Monoalkyl [4,4] DM-[4,5](2 μM) 2790 (100%) no N-demethylation no exposed primary aminopropylterminal segment DE-[4,5] (100 μM) 4215 (100%) no N-deethylation ″DP-[4,5] (100 μM) 1325 (69.7%) N10-MP-[5,4]* 576 (30.3%) ″ noelaboration of N-Monoalkyl [5,4] DM-[6,5] (15 μM) 2560 (100%) noN-demethylation no exposed primary aminopropyl terminal segment DE-[6,6](75 μM) 1585 (100%) no N-deethylation ″ DP-[6,5] (500 μM) 1300 (100%) noN-depropylation ″

[0097] In order to assure that the observation was not some artifact ofthe experimental conditions, it was assessed whether or not componentsof the culture media itself were responsible for dealkylation (Table 5).Fetal bovine serum (FBS), for example, is well known to contain amineoxidases [Morgan, “Polyamine Oxidases and Oxidized Polyamines,” Chapter13 in The Physiology of Polyamines, Vol. I; Bachrach et al, eds., CRCPress: Boca Raton, Fla. (1989), pages 203-229]. Indeed, 1 mMaminoguanidine, an inhibitor [Gahl et al, “Reversal by Aminoguanidine ofthe Inhibition of Proliferation of Human Fibroblasts by Spermidine andSpermine,” Chem.-Biol. Interactions, Vol. 22, pages 91-98 (1978)] ofbovine serum amine oxidase present in the standard L1210 cell culturemedia, did not totally eliminate such FBS-related amine oxidaseactivity. When the “complete” RPMI-40 medium containing FBS and 1 mMaminoguanidine was incubated in the presence of 100 or 500 μM DPNSPD, asmall amount (<3%) of the DPNSPD was metabolized to MPNSPD in theabsence of L1210 cells. This corresponds to a comparatively lowextracellular concentration of MPNSPD (˜3 μM) and, given its relativelypoor affinity (K_(i)=33 μM) for the polyamine transport apparatus,argues against the extracellular medium as a major source of the highlevels of MPNSPD (264 μM) seen intracellularly. This conclusion isfurther supported by experiments which partially or totally eliminatethe source of extracellular metabolism. For example, when FBS wasreplaced with either NuSerum, a semi-synthetic substitute, or purifiedbovine serum albumin, a high level of intracellular metabolite (50% ofparent analogue, Table 5) was still observed. The chelatorbathophenanthroline disulfonic acid is a well-known inhibitor of theCu-dependent amine oxidases present in plasma [Frieden, “Complex Copperof Nature,” Metamorphosis, A Problem in Developmental Biology, 2nd ed.,Gibert et al, eds., Plenum Press: New York, N.Y., pages 478-483 (1981)]and, given its comparatively high MW and anionic charge, does not crossthe cell membrane [Alcain et al, “Iron Reverses Impermeable ChelatorInhibition of DNA Synthesis in CCl 39 cells,” Proc. Natl. Acad. Sci.U.S.A., Vol. 91, pages 7903-7906 (1994); and Glahn et al,“Bathophenanthroline Disulfonic Acid and Sodium Dithionite EffectivelyRemove Surface-Bound Iron from CaCO₂ Cell Monolayers,” J. Nutr., Vol.125, pages 1833-1840 (1995)]. As expected, bathophenanthrolinedisulfonic acid completely abolished the ability of RPMI-40+10% FBS toconvert DPNSPD to MPNSPD. However, when cells were grown inRPMI-containing FBS and bathophenanthroline disulfonic acid, highintracellular concentrations of MPNSPD corresponding to ca. 50% of theintracellular DPNSPD content were observed. These results are in keepingwith the idea that the dealkylation indeed takes place within L1210cells. TABLE 5 METABOLISM OF DPNSPD IN DIFFERENT CULTURE SYSTEMSMetabolites Assay # Experiment Treatments (% of DPNSPD) 1 FBS^(a) +L1210 (48 hours) MPNSPD (50%) 2 NuSerum^(a) + L1210 (48 hours) MPNSPD(50%) 3 FBS (48 hours) MPNSPD (3%) 4 Albumin^(b) + L1210 (4 hours)MPNSPD (50%) 5 FBS + bathophenanthroline disul- MPNSPD (0%) fonic acid(0.1 mM) (48 hours) 6 FBS + L1210 + bathophenanthroline MPNSPD (50%)disulfonic acid (0.1 mM) (48 hours)

[0098] Assured that what was being seen were the results ofintracellular metabolic transformation of bisalkylated triamines, anexamination of the influence of polyamine analogue structure on themetabolite pattern observed in L1210 cells was undertaken. These resultsare detailed in Table 4 for the bisalkyl triamines and a number of theirprimary metabolites. Several representative tetraamine analogues arealso included to demonstrate their similar metabolic fate to thecorresponding triamines. Note, too, that the structures are depictedwith the polyamine backbone described as Arabic numerals separated bycommas so that the numeral represents the number of methylenes in thelinear alkane sections separating amine centers, thus [3,3]=NSPD,[3,4,3]=SPM, [4]=putrescine, and so forth.

[0099] Three types of metabolic transformations explain the particularpatterns observed (FIG. 7). First, bisalkyl polyamines must undergoN-dealkylation before any further metabolism can occur. If thisN-dealkylation results in exposure of a primary aminopropyl segment, theprimary metabolite(s) may undergo deaminopropylation by the SSAT/PAOpolyamine degradation pathway. If this N-dealkylation results inexposure of a primary aminobutyl segment, then the triamine mightundergo elaboration into a tetraamine by serving as a substrate forspermine synthase, which anneals an aminopropyl segment derived fromS-adenosylmethionine (AdoMet) to the free aminobutyl end of themolecule. Below, the evidence as revealed in the metabolite patternsthat support these three types of metabolic transformations is detailed,along with comments on the implications these data have with respect tothe structural requirements of the corresponding enzyme systems in vivo.

[0100] A careful inspection of chromatograms from cells treated withDM-[3,3], DM-[3,4], DM-[4,4], DM-[4,5] and DM-[5,5] revealed noN-demethylation (Table 4). The dimethyl tetraamines DM-[3,3,3],DM-[3,4,3] and DM-[4,4,4] also showed no evidence of N-dealkylation(data not shown), and the unsymmetric tetraamine MM-[3,3,3] is onlymetabolized by deaminopropylation at the primary amine terminus end ofthe molecule.

[0101] Treatment of cells with DE-[3,3] or the corresponding tetraamine,DE-[3,3,3], each resulted in the monodeethylated metabolite, ME-[3,3] orME-[3,3,3], respectively, in similar amounts: 4.7% on a mole percentbasis of the total (parent drug +identified metabolites) in the cell.Interestingly, cells treated with the unsymmetric DE-[3,4] contain eachof the two possible monodeethylated metabolites, N1-ME-[3,4] andN8-ME-[4,3] with the total amount representing about 4% of the drug inthe cell. Among the diethylated triamine analogues, only DE-[3,3] andDE-[3,4] showed N-deethylated metabolite(s); the analogues with longerbackbones, i.e., DE-[4,4], DE-[4,5] and DE-[5,5], do not showsignificant N-deethylation at all.

[0102] Of the five different dipropyl triamines which were evaluated,DP-[3,3], DP-[3,4], DP-[4,4], DP-[4,5] and DP-[5,5], all but DP-[5,5]showed significant N-depropylation. As suggested from the quantity ofanalogue present in cells as monodealkylated metabolite (Table 4),N-depropylation in general occurs to a greater extent thanN-deethylation. For example, in L1210 cells treated with DP-[3,3], 57.7%is present as the parent drug, DP-[3,3], 30.6% as the mono-N-dealkylatedmetabolite, MP-[3,3], 9.1% as the di-N-dealkylated metabolite, [3,3],and 2.6% as the secondary metabolite, MP-[3], formed bydeaminopropylation of MP-[3,3]. The same general pattern holds for cellstreated with the corresponding tetraamine, DP-[3,3,3], where 14.0% ofthe total is present as MP-[3,3,3] and 7.8% as MP-[3,3] formed bysecondary deaminopropylation of MP-[3,3,3]. Cells treated with thecorresponding diethyl analogues contain substantially lower amounts ofmetabolites by comparison so that 90.7% of DE-[3,3] or 95.3% ofDE-[3,3,3] is present as the unmetabolized parent compound.

[0103] The dipropyl triamine with the shortest backbone DP-[3,3] seemedmost sensitive to metabolism with MP-[3,3] representing 31% of the totaldrug. With DP-[3,4], both possible monoalkylated products N1-MP-[3,4]and N8-MP-[4,3] were detected at levels corresponding to 4.6% and 5.3%,respectively, of the total drug in the cell. Cells exposed to DP-[4,4]contained the mono-N-dealkylated metabolite, MP-[4,4], representing12.3% of the total drug in the cell. Interestingly, only one of the twopossible N-dealkylated metabolites was apparent in cells treated withthe unsymmetric triamine DP-[4,5], and this MP-[5,4] metaboliterepresented 32.9% of the total drug in the cell. When DP-[5,5] wasevaluated, no metabolic products were found, suggesting that theaminobutyl end of DP-[4,5] system was selectively dealkylated to formthe monodealkylated metabolite, N10-MP-[5,4].

[0104] If mono-N-dealkylation exposes a primary aminopropyl terminus,this compound is subject to further metabolism by the SSAT/PAO systempresent in all cells. First, SSAT acetylates the exposed primary amineend, then PAO oxidatively deaminates at the interior secondary aminonitrogen of the acetamidopropylamine segment to give acetamidopropanal,i.e., net deaminopropylation of the substrate. PAO activelydeaminopropylates N¹-acetylspermine and N¹-acetylspermidine, the nativesubstrates, but does not recognize the acetamidobutyl segment ofN8-acetylspermidine or N-acetylputrescine as substrate. Table 4demonstrates that, in L1210 cells, there is a strict adherence to thisspecificity for a primary aminopropyl segment for further metabolism ofthe monoalkylated triamines, i.e., only examples of deaminopropylationare observed. For example, the tetraamine MM-[3,3,3] shows a substantialamount of the deaminopropylation metabolite, MM-[3,3], representing16.5% of the total drug in the cell and even some MM-[3] (0.6%), theproduct of deaminopropylation of MM-[3,3]. No examples ofdeaminobutylation are seen, e.g., N8-alkyl-[4,3], monoalkyl-[4,4] andmonoalkyl-[5,4] do not give rise to such metabolites. In the case ofcells treated with ME-[3,3] or MP-[3,3], both N-dealkylation anddeaminopropylation are available paths of primary metabolism. Thedeaminopropylation metabolite, ME-[3], represented 21.3% of the totaldrug in the ME-[3,3] treated cells compared to only 0.5% for theN-deethylation product, [3,3]. In contrast, the N-depropylation product,[3,3] (4.5%), predominated compared to the deaminopropylationmetabolite, MP-[3], in MP-[3,3] treated cells.

[0105] In cells treated with the N¹-monoalkylated spermidines,N1-ME-[3,4] or N1-MP-[3,4], peaks corresponding to the respectivetetraamines ME-[3,4,3] (3.7% of total drug in cell) and MP-[3,4,3] (6.1%of total drug in cell) were observed in the HPLC chromatograms of thedansylated cell extract (Table 4). In the case of cells containingsubstantial amounts of triamine analogues with a free primaryaminopropyl end (i.e., ME-[3,3], MP-[3,3], N8-ME-[4,3] and N8-MP-[4,3]),no evidence of a tetraamine elaboration metabolite was observed. only inthose cases where a free aminobutyl end was available on a spermidine,[3,4], backbone was a tetraamine metabolite produced. No such metabolitewas produced from triamines with a free aminobutyl end on a longerbackbone (i.e., MP-[4,4] or N10-MP-[5,4]).

[0106] Thus, it is likely that at least two of the pathways responsiblefor metabolic transformation involve enzymes of the polyamine metaboliccycle present in all cells. Spermine synthase is responsible forelaboration of a spermidine analogue to the correspondingN-alkylspermine by annealing an aminopropyl segment to an exposedprimary aminobutyl end of the triamine. The deaminopropylation observedin L1210 cells treated with triamine and tetraamine analogues is readilyexplained as a consequence of action by the SSAT/PAO polyaminedegradative enzymes. The possibility that the N-dealkylation steprequired for further metabolic transformation of bis-alkylpolyamines mayalso involve PAO is an interesting question raised by the metabolicpatterns observed.

[0107] N-Dealkylation of analogues with a hydrophobic segment shorterthan N-propyl appears to occur much less efficiently in the case ofN-ethyl, or not at all in the case of N-methyl. Among the reported amineoxidases, polyamine oxidase (PAO) is the only one which usually attacksat a secondary amine center, three hydrophobic methylene carbonsinternal to the neutral N¹-acetamido nitrogen terminus ofN¹-acetylspermidine, for example. The corresponding acetamidobutylsegment of N⁸-acetylspermidine is not recognized and, therefore, notdeaminobutylated.

[0108] Conservation of Charge.

[0109] In two earlier studies, it was noted that there was aconservation of charge with respect to the total tetraamine cationicpicoequivalence in the cell [Bergeron et al, Cancer Res., Vol. 49,supra; and Porter et al, Cancer Res., Vol. 51, supra]. For example, if,after 24 hours of exposure to an alkylated polyamine, each of theequivalent concentrations associated with charge on the amines of boththe analogues and natural polyamine is added together, the numbers arefairly constant. For example, each picoequivalent of putrescine isassociated with two picoequivalents of cationic charge, eachpicoequivalent of spermidine or its analogues with three, and eachpicoequivalent of spermine with four. In order to maintain this balanceof charge, the cell processes the natural polyamines, e.g., exports themas it incorporates the analogues. The maintenance of total cellularcharge holds for all of the triamines examined, except the 5,5 triamines(Table 6). The implication is that the cell will not incorporate theanalogue beyond a point where the charge balance is disrupted, at whichtime cell death may occur. In the case of the tetraamines, theconservation of charge behavior seems to hold for 24 hours, but erodesafter a period of time [Bergeron et al, Cancer Res., Vol. 49, supra].With the triamines, the conservation of charge continues even at 48hours. TABLE 6 SUMMATION OF INTRACELLULAR LEVELS OF ANALOGUES ANDPOLYAMINES ANALYZED FOR AMINE EQUIVALENCE AFTER EXPOSURE TO POLYAMINEANALOGUES Polyamine Picoequivalents of Amine/ Average ± StandardAnalogues 10⁶ cells (× 10³) Deviation Control Cell 13.21 2 DMNSPD 18.404 DENSPD 13.70 5 MPNSPD 17.71 6 DPNSPD 15.01 16.21 ± 2.22 8 DMSPD 16.099 MESPD(N1) 16.99 10 MESPD(N8) 17.99 11 DESPD 14.05 12 MPSPD(N1) 18.2513 MPSPD(N8) 19.59 14 DPSPD 15.33 16.90 ± 1.89 16 DMHSPD 20.34 17 DEHSPD16.92 18 MPHSPD 20.55 19 DPHSPD 15.99 18.45 ± 2.34 21 DM(4,5) 20.81 22DE(4,5) 14.59 23 DP(4,5) 15.15 16.85 ± 3.44 24 DM(5,5) 15.5 25 DE(5,5)9.01 26 DP(5,5) 13.91 12.81 ± 3.38 All Analogues Mean 16.47 ± 2.08 #bytwo and analogue by three. The typical control values in nmol/millionL1210 cells are PUT = 0.260 ± 0.059, SPD = 3.354 ± 0.361, SPM = 0.658 ±0.119.

[0110] Acute and Chronic Toxicity of Triamines.

[0111] In early studies of polyamine toxicity in laboratory animals,triamines were found less toxic than tetraamines. Spermidine wasapproximately one-twentieth as nephrotoxic as spermine, and putrescinewas the least toxic [Tabor et al, “Pharmacology of Spermine andSpermidine. Some Effects on Animals and Bacteria,” J. Pharmacol. Exp.Ther., Vol. 116, pages 139-155 (1956); and Shaw, “Some PharmacologicalProperties of the Polyamine Spermine and Spermidine - a Re-appraisal,”Arch. Int. Pharmacodyn. Ther., Vol. 198, pages 36-48 (1972)].

[0112] In the current study, the acute toxicity of six analogues and thechronic toxicity of two triamines were measured (Table 7). The value ofall polyamine LD₅₀s are shown in both mg/kg and mmol/kg for comparison.For acute toxicities, the polyamine analogues were administered as asingle i.p. injection to groups of five or six animals at each dose. Theanimals were scored two hours after administration of the drug. It isclear that the acute LD₅₀s for triamine analogues are approximatelytwice the acute LD₅₀s for the corresponding tetraamine analogues.

[0113] In the chronic toxicity regimen, mice were administered thepolyamine analogue in three doses per day (t.i.d.) for six days for atotal of eighteen injections per animal and observed for 10 days afterthe final dose for lethalities. The most active triamine DE(4,5) againstL1210 cells in vitro and the spermidine analogue DE(3,4) demonstratedmuch less toxicity in mice than the related tetraamines DE(4,5,4),DE(5,4,5) and DE(3,4,3). In the early study of tetraamines, apreliminary investigation suggested a direct ratio relationship betweenthe IC₅₀ and the chronic LD₅₀ values [Bergeron et al, J. Med. Chem.,Vol. 37, supra]. However, in the triamine systems, the 96-hour IC₅₀values of DE(4,5) suggested that this triamine should be about 5 timesless toxic than corresponding tetraamine analogue, DE(4,5,4), and sixtimes less toxic than DE(5,4,5) (Table 1), but, in fact, they are abouteight-fold less toxic than the DE(4,5,4) and greater than ten-fold lesstoxic than DE(5,4,5) (Table 7). A similar difference is also observed inthe chronic toxicity of DE(3,4). The ratio of the triamine to thetetraamine 96-hour IC₅₀s suggests that DE(3,4) should be approximatelyfour times less toxic than DE(3,4,3), but, in fact, DE(3,4) is aboutfive times less toxic than DE(3,4,3) in vivo. These results suggest apotential widening of the therapeutic window, which renders the triamineanalogues as promising anti-neoplastics of lower toxicity and encouragesfurther pursuit of animal studies. TABLE 7 COMPARISON OF THE ACUTE ANDCHRONIC TOXICITY OF TETRAAMINE AND TRIAMINE ANALOGUES ON MICETETRAAMINES TRIAMINES Acute^(a) LD₅₀ mg/kg Chronic^(b) LD₅₀ mg/kg- AcuteLD₅₀ mg/kg Chronic LD₅₀ mg/kg- Compound (mmol/kg) day (mmol/kg-day)Compound (mmol/kg) day (mmol/kg-day) DE-[3,4,3] 340 (0.842) 87^(e)(0.215) DE-[3,4] >650^(d) (>3.22) 426 (1.37) DE-[4,5,4] 285 (0.638) 48(0.104) DE-[4,5] 555^(e) (1.64) 375 (1.11) DE-[5,4,5] 195 (0.424) 36(0.078) DE-[5,5] 500 (1.42) nd

[0114] The study serves to define the similarities and differencesbetween triamines and tetraamine analogue antineoplastics. With bothtetraamine and triamine analogues, K_(i) values are sensitive to thesize of the terminal substituents and the length of the backbone. Thisis illustrated for triamines in FIG. 8. Generally, the larger theterminal substituent, the more poorly the analogues are transported. Inthe triamine family, spermidine analogues are the best transportcompetitors. Interestingly, the (3,3) and (5,5) triamine analogues aremost sensitive to N-terminal substituent changes. With regards touptake, the triamines are more effectively accumulated in L1210 cellsthan the corresponding tetraamines. Once in the cell, tetraamineanalogues have a greater impact on lowering overall polyamine pools;however, the triamines are more selective at reducing spermidine. Thetotal intracellular charge in picoequivalents associated withpolyamines, both native and analogues, is maintained by cells exposed toboth tetraamines and triamines. However, cells treated with triaminesare able to maintain this charge balance for a more prolonged period oftime. Both tetraamine and triamine analogues, except for DENSPD andDE(4,5), reduce ODC more effectively than AdoMetDC activity, andtetraamines are more active at this. It was demonstrated that triamineanalogue dealkylation was very specific for triamines with backbones ofless than or equal to four methylenes and most effective for triaminesand tetraamines with N^(α),N^(ω)-dipropyl substituents.

[0115] The tetraamine analogues are uniformly more active against L1210cells than their triamine counterparts. With both the triamine andtetraamine analogues the compounds' IC₅₀ values are also sensitive tothe size of the terminal substituent and the length of the backbone.However, the overall length between the terminal nitrogens is the mostcritical issue in assessing this activity; FIG. 9a illustrates thetriamine case. When comparing N¹- with N⁸-monoalkylspermidines, the N¹compounds, both ethyl and propyl were more active than the N⁸ compound.The fact that the N¹ compounds are elaborated by the cell to thecorresponding and more active N¹-alkylspermines is in keeping with thisobservation. It is recalled that the N⁸-alkylspermidines cannot be andare not further processed in the polyamine biosynthetic network. Whilethe optimum length for the tetraamine activity has not yet beendetermined (FIG. 9b), evidence would suggest that the optimum length forthe triamines has been determined, as seen in the terminally dialkylated(4,5)-methylene backbone series.

[0116] The triamine analogues are less toxic than the correspondingtetraamines. Furthermore, and most important when comparing the ratio ofthe 96-hour IC₅₀/chronic LD₅₀ values of the two triamines, DE(3,4) andDE(4,5), with the corresponding tetraamines, a kind of therapeuticwindow, the triamines appear more favorable. This is a critical issue inthe choice of the best polyamine therapeutic.

[0117] The invention is illustrated by the following non-limitingexamples wherein parenthetical reference numerals correspond to those inSchemes 1-5.

[0118] MENSPD (3) [Bergeron et al, Drug Metab. Dispos., Vol. 23, supra]and tetraamine analogues [Bergeron et al, J. Med. Chem., Vol. 31, supra;and Bergeron et al, J. Med. Chem., Vol. 37, supra], except for DPNSPMand DE(5,4,5), were previously synthesized. N¹- and N⁸-Acetylspermidinedihydrochlorides were purchased. N—(3-aminopropyl)-1,3-propanediamine(1) was converted to its trihydrochloride salt and recrystallized fromaqueous ethanol. Sodium hydride reactions were run in distilled DMFunder an inert atmosphere. THF was distilled from sodium andbenzophenone. Fisher Optima grade solvents were routinely used, andorganic extracts were dried with sodium sulfate. Silica gel 32-63 (40 μM“flash”) was used for flash column chromatography. Melting points weredetermined on a Fisher-Johns melting point apparatus and areuncorrected. Proton NMR spectra were run at 90 or 300 MHz in CDCl₃ (notindicated) or D₂O with chemical shifts in parts per million downfieldfrom tetramethylsilane or 3-(trimethylsilyl)propionic-2,2,3,3-d₄ acid,sodium salt, respectively. Coupling constants (J) are in hertz. FAB massspectra were run in a glycerol/trifluoroacetic acid matrix. Elementalanalyses were also performed.

[0119] Cell culture materials, RPMI-1640 medium, fetal bovine serum,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and3-(N-morpholino)propanesulfonic acid (MOPS) were purchased. Cell numberswere determined by electronic particle analysis (Coulter Counter, ModelZF). The solid phase extraction columns (SPE-3 mL/500 mg) were used.Murine L1210 leukemia cells were obtained from the American Type TissueCorporation.

[0120] [³H]Spermidine for uptake determinations and acetyl coenzyme A(acetyl-1-¹⁴C) were purchased. L-[Carboxyl-¹⁴C]-ornithine andS-adenosyl-L-[carboxyl-¹⁴C]methionine for enzyme assays were alsopurchased.

[0121] Cell Culture and IC₅₀ Determination.

[0122] Murine L1210 leukemia cells (ATCC CCL 219) were maintained inlogarithmic growth in RPMI-1640 medium containing 10% fetal calf serumor a semi-synthetic equivalent, NuSerum, 2% HEPES-MOPS buffer and 1 mMaminoguanidine. The IC₅₀s, the concentration of compound which reducescell growth to 50% of untreated control cell growth, was determinedafter 48 hours and 96 hours of exposure to polyamine analogue asdetailed elsewhere [Bergeron et al, J. Med. Chem., Vol. 37, supra].

[0123] Polyamine Pool Analysis.

[0124] L1210 cells in logarithmic growth were treated with polyamineanalogue at the concentrations indicated in Table 4 for 48 hours. Thecells were washed twice with cold RPMI-1640, and the pellet was treatedwith 0.6 N HClO₄ (1 ml/10⁷ cells). Polyamine contents of the perchloricacid extracts were quantitated by HPLC of the DANSYL derivatives[Bergeron et al, J. Med. Chem., Vol. 37, supra].

[0125] Uptake Determinations.

[0126] The polyamine derivatives were studied for their ability tocompete with [³H]-SPD for uptake into L1210 cells [Bergeron et al, J.Med. Chem., Vol. 37, supra]. Lineweaver-Burk plots indicated a simplecompetitive inhibition with respect to SPD.

[0127] Enzyme Assays.

[0128] ODC and AdoMetDC activities were determined as ¹⁴CO₂ releasedfrom [¹⁴C]-carboxyl-labeled L-ornithine [Seely et al, “OrnithineDecarboxylase (Mouse Kidney),” Methods Enzymol., Vol. 94, pages 158-161(1983)] or S-adenosyl-L-methionine [Pegg et al, “S-AdenosylmethionineDecarboxylase (Rat Liver),” Methods Enzymol., Vol. 94, pages 234-239(1983)], respectively. Included in each assay were untreated L1210 cellsas controls, as well as cells treated with DEHSPM, a drug having a knownreproducible effect on each enzyme, as positive controls.

[0129] Spermidine/spermine N¹-acetyltransferase activity was based onquantitation of [¹⁴C]-N¹-acetylspermidine formed by acetylation of SPDwith [¹⁴C] acetyl coenzyme A according to the method of Libby et al[Biochem. Pharmacol., Vol. 38, supra]. Cells treated with DENSPM werepositive controls.

[0130] Toxicity in Mice.

[0131] Acute and chronic toxicities were assessed in 10-12 week old CD-1female mice from Harlan Sprague-Dawley. For acute toxicities, thepolyamine analogues were administered in a single i.p. injection togroups of five or six animals at each dose. The animals were scored twohours after administration of the dose. All survivors were furtherobserved for 10 days to assess late onset of toxicity from the singleacute dose. In the chronic toxicity regimen, mice were administeredpolyamine analogue in three i.p. doses per day (t.i.d.) for six days,for a total of eighteen doses per animal. Appetite, weight and overallappearance were monitored daily. Animals were observed for 10 daysfollowing the final dose, at which time the final score was registered.At least three test groups of 5-6 animals each, representing threedifferent dose levels, were evaluated for each analogue tested. Thesedose levels were chosen so that at least two groups presented withlethalities, one with a high fraction of lethalities (>0.50, but <1.00).

EXAMPLE 1

[0132] N¹,N⁴,N⁷-Tris (mesitylenesulfonyl)-N¹,N⁷-dimethylnorspermidine(43).

[0133] NaH (60% in oil, 0.44 g, 11 mmol) was added to a solution of 30[Bergeron et al, Drug Metab. Dispos., Vol. 23, supra] (3.39 g, 5 mmol)in DMF (70 mL) at 0° C. After hydrogen evolution ceased (30 minutes),iodomethane (1.63 g, 11.5 mmol) was slowly added to the mixture. Afterstirring for 12 hours at room temperature, the reaction mixture wasquenched with distilled water (10 mL). The solvents were removed underhigh vacuum, and the residue was combined with H₂O (30 mL) and extractedwith CHCl₃ (4×40 mL). The organic portion was washed with brine (80 mL)and evaporated by rotary evaporation. Purification by columnchromatography (8:1 toluene/EtOAc) gave 2.47 g (70%) of 43 as an oil:NMR δ 1.73 (quintet, 4H), 2.29 (s, 6H), 2.54-2.55 (2 s, 18H), 2.60 (s,6H), 2.99-3.06 (2 t, 8H, J=7), 6.94 (s, 6H). Anal. (C₃₅H₅₁N₃O₆S₃) C, H,N.

EXAMPLE 2

[0134] N¹,N⁷-Dimethylnorspermidine Trihydrochloride (2).

[0135] HBr (30% in HOAc, 30 mL) was added slowly to a mixture of 43(2.13 g, 3.02 mmol) and phenol (12.27 g, 0.13 mol) in CH₂Cl₂ at 0° C.After stirring for 1 day at room temperature, H₂O (20 mL) was added tothe reaction mixture followed by extraction with CH₂Cl₂ (3×30 mL). Theaqueous portion was concentrated under high vacuum, and the residue wasbasified to pH 14 with 1 N NaOH (4 mL) and 19 N NaOH (2 mL) andextracted with CHCl₃ (14×10 mL). The organic extracts were concentrated,and the residue was taken up in absolute EtOH (40 mL) and acidified withconcentrated HCl (2 mL). After the solvents were removed, the solid wasrecrystallized from aqueous EtOH to generate 0.298 g (37%) of 2 asplates: NMR (D₂O) δ 2.08-2.19 (m, 4H), 2.75 (s, 6H), 3.13-3.22 (m, 8H).Anal. (C₈H₂₄Cl₃N₃) C, H, N.

EXAMPLE 3

[0136] N¹,N⁴,N⁷-Tris (mesitylenesulfonyl)-N¹,N⁷-diethylnorspermidine(44).

[0137] NaH (60%, 9.0 g, 0.23 mol), 30 [Bergeron et al, Drug Metab.Dispos., Vol. 23, supra] (70.0 g, 0.103 mol), and iodoethane (19 mL,0.24 mol) in DMF (400 mL) were reacted and worked up by the method of43. Column chromatography (1:4 EtOAc/pet ether) afforded 63.7 g (84%) of44 as a viscous oil: NMR δ 0.97 (t, 6H, J=7), 1.60-1.72 (m, 4H), 2.29(s, 9 H), 2.54 and 2.55 (2 s, 18H), 2.95-3.15 (m, 12H), 6.92 (s, 6H).Anal. (C₃₇H₅₅N₃O₆S₃) C, H, N. A sample was recrystallized from EtOAc/petether, mp 97° C.

EXAMPLE 4

[0138] N¹,N⁷-Diethylnorspermidine Trihydrochloride (4).

[0139] HBr (30% in HOAc, 300 mL), 44 (23.0 g, 30.7 mmol), and phenol(116 g, 1.23 mol) in CH₂Cl₂ (300 mL) were reacted, and product wasisolated using the procedure of 2 to give 6.45 g (71%) of 4 as colorlessplates: NMR (D₂O) δ 1.30 (t, 6H, J=7), 2.05-2.18 (m, 4H), 3.08-3.22 (m,12H). Anal. (C₁₀H₂₈Cl₃N₃) C, H, N.

EXAMPLE 5

[0140] N¹-Propyl-N¹,N⁴,N⁷-tris(mesitylenesulfonyl)norspermidine (45).

[0141] NaH (60%, 0.52 g, 13 mmol), 30 (2.57 g, 3.8 mmol), and1-iodopropane (0.46 mL, 4.7 mmol) in DMF were reacted and worked up bythe method of 43. Column chromatography (3:1 hexane/EtOAc) afforded 1.16g (23%) of 45 as an oil: NMR 66 0.66 (t, 3H, J=7), 1.23-1.31 (m, 2H),1.58-1.62 (m, 4H), 2.26-2.27 (2 s, 9H), 2.51-2.52 (2 s, 12H), 2.59 (s,6H), 2.82-2.99 (m, 8H), 3.21 (t, 2H, J=7), 4.85 (br t, 1H), 6.89-6.92(m, 6H). Anal. (C₃₆H₅₃N₃O₆S₃) C, H, N.

EXAMPLE 6

[0142] N¹-Propylnorspermidine Trihydrochloride (5).

[0143] HBr (30% in HOAc, 30 mL), 45 (1.14 g, 1.58 mmol), and phenol (6.4g) in CH₂Cl₂ were reacted, and product was isolated using the procedureof 2 to give 87 mg (20%) of 5 as crystals: NMR (D₂O) δ 0.98 (t, 3H,J=7), 1.67-1.75 (m, 2H), 2.07-2.16 (m, 4H), 3.01-3.21 (m, 10H). Anal.(C₉H₂₆Cl₃N₃) C, H, N.

EXAMPLE 7

[0144] N¹,N⁷-Dipropyl-N¹,N⁴,N⁷-tris(mesitylenesulfonyl)-norspermidine(46).

[0145] NaH (60%, 0.44 g, 11 mmol), 30 [Bergeron et al, Drug Metab.Dispos., Vol. 23, supra] (3.39 g, 5 mmol), and 1-iodopropane (1.95 g,11.5 mmol) in DMF (70 mL) were reacted and worked up by the method of43. Column chromatography (3:1 hexane/EtOAc) afforded 3.54 g (93%) of 46as an oil: NMR δ 0.7 (s, 6H), 1.20-1.65 (m, 8H), 2.25 (s, 9H), 2.50 (s,18H), 2.80-3.05 (m, 12H), 6.87 (s, 6H). Anal. (C₃₉H₅₉N₃O₆S₃) C, H, N.

EXAMPLE 8

[0146] N¹,N⁷-Dipropylnorspermidine Trihydrochloride (6).

[0147] HBr (30% in HOAc, 80 mL), 46 (3.475 g, 4.57 mmol), and phenol(15.8 g) in CH₂Cl₂ (30 mL) were reacted, and product was isolated by theprocedure of 2 to provide 1.26 g (85%) of 6 as plates: NMR (D₂O) δ 0.87(t, 6H, J 7), 1.60 (m, 4H), 2.01 (m, 4H), 2.93 (t, 4H, J=7), 3.06 (m,8H). Anal. (C₁₂H₃₂Cl₃N₃) C, H, N.

EXAMPLE 9

[0148] N¹,N⁴,N⁸-Tri (mesitylenesulfonyl) spermidine (31).

[0149] Mesitylenesulfonyl chloride (6.87 g, 31.4 mmol) in CH Cl₂ (30 mL)was added to spermidine trihydrochloride (2.5 g, 9.8 mmol) in 1 N NaOH(35 mL) at 0° C., and the mixture was efficiently stirred at roomtemperature overnight. The layers were separated, and the aqueous phasewas extracted with CHCl₃ (3×50 mL). The organic phase was the washedwith brine (100 mL), evaporated, and purified by column chromatography(4:3 hexane/EtOAc) to give 3.73 g (55%) of 31 as a white foam: NMR 671.30 (m, 2H), 1.44 (m, 2H), 1.66 (m, 2H), 2.30 (s, 9H), 2.46 (s, 6H),2.60 (s, 12H), 2.76 (q, 2H), 2.84 (q, 2H), 3.04 (t, 2H, J=7), 3.24 (t,2H, J=7), 4.56 (br t, 1H), 4.92 (br t, 1H), 6.90 (s, 2H), 6.95 (s, 4H).Anal. (C₃₄H₄₉N₃O₆S₃) C, H, N.

EXAMPLE 10

[0150] N¹,N⁸-Dimethyl-N¹,N⁴,N⁸-tris (mesitylenesulfonyl) spermidine(47).

[0151] NaH (60%, 0.41 g, 10 mmol), 31 (2.15 g, 3.11 mmol), andiodomethane (0.62 mL, 10 mmol) in DMF (60 mL) were reacted and worked upas was 43. Column chromatography (5:3 hexane/EtOAc) furnished 2.24 g(100%) of 47 as an oil: NMR δ 1.39-1.43 (m, 4H), 1.69-1.78 (m, 2H), 2.28(s, 3H), 2.30 (s, 6H), 2.55 (s, 12H), 2.57 (s, 6H), 2.60 (s, 3H), 2.62(s, 3H), 2.96-3.13 (m, 8H).

EXAMPLE 11

[0152] N¹,N⁸-Dimethylspermidine Trihydrochloride (8).

[0153] HBr (30% in HOAc, 60 mL), 47 (2.24 g, 3.11 mmol), and phenol(12.3 g) in CH₂Cl₂ (30 mL) were reacted, and product was isolated by theprocedure of 2 to give 0.658 g (75%) of 8 as crystals: NMR (D₂O) δ1.77-1.82 (m, 4H), 2.06-2.18 (m, 2H), 2.73 (s, 3H), 2.75 (s, 3H),3.06-3.19 (m, 8H). Anal. (C₉H₂₆Cl₃N₃) C, H, N.

EXAMPLE 12

[0154] N¹,N⁸-Diethyl-N¹,N⁴,N⁸-tris(mesitylenesulfonyl)spermidine (48).

[0155] NaH (80%, 0.68 g, 23 mmol), 31 (7.06 g, 10.2 mmol), andiodoethane (2.5 mL, 31 mmol) in DMF (75 mL) were combined as in 43. Themixture was heated at 65° C. for 12 hours, cooled and cautiouslyquenched with water (70 mL) and brine (100 mL), followed by extractionwith EtOAc (5×100 mL). Combined organic extracts were washed with 100 mLof 1% Na₂SO₃, H₂O (2×), and brine. The solvents were removed, and theresidue was purified by column chromatography (4.5% EtOAc/CH₂Cl₂) toproduce 7.21 g (94%) of 48 as an oil: NMR δ 0.8-1.8 (m, 12H), 2.28 (s,9H), 2.54 (s, 18H), 2.8-3.3 (m, 12H), 6.90 (s, 6H). Anal. (C₃₈H₅₇N₃O₆S₃)C, H, N.

EXAMPLE 13

[0156] N¹,N⁸-Diethylspermidine Trihydrochloride (11). HBr

[0157] (30% in HOAc, 150 mL), 48 (7.16 g, 9.57 mmol), and phenol (28 g,0.30 mol) in CH₂Cl₂ (125 mL) were reacted, and product was isolatedutilizing the procedure of 2 to give 2.17 g (73%) of 11 as white plates:NMR (D₂O) δ 1.28 and 1.30 (2 t, 6H, J=7), 1.73-1.85 (m, 4H), 2.06-2.18(m, 2H), 3.04-3.21 (m, 12 H). Anal. (C₁₁H₃₀Cl₃N₃) C, H, N.

EXAMPLE 14

[0158] N¹,N⁸-Dipropyl-N¹,N⁴,N⁸-tris(mesitylenesulfonyl)-spermidine (49).

[0159] NaH (80%, 0.80 g, 27 mmol) was added to 31 (8.13 g, 11.7 mmol) inDMF (75 mL) at 0° C. The mixture was stirred at room temperature for 1hour, and 1-iodopropane (3.5 mL, 36 mmol) was added by syringe. Themixture was stirred at 80° C. for 12 hours and worked up as was 48.Purification by column chromatography (3.5% EtOAc/CH₂Cl₂) resulted in8.42 g (93%) of 49 as an oil: NMR δ 0.55-1.72 (m, 16H), 2.25 (s, 9 H),2.50 (s, 18H), 2.7-3.3 (m, 12H), 6.87 (s, 6H). Anal. (C₄₀H₆₁N₃O₆S₃) C,H, N.

EXAMPLE 15

[0160] N¹,N⁸-Dipropylspermidine Trihydrochloride (14).

[0161] HBr (30% in HOAc, 150 mL), 49 (8.32 g, 10.7 mmol), and phenol (28g, 0.29 mol) in CH₂Cl₂ (125 mL) were reacted, and product was isolatedby the procedure of 2 to produce 2.58 g (71%) of 14 as white plates: NMR(D₂O) δ 0.97 and 0.98 (2 t, 6H, J=7), 1.63-1.83 (m, 8H), 2.06-2.19 (m,2H), 2.97-3.21 (m, 12H). Anal. (C₁₃H₃₄Cl₃N₃) C, H, N.

EXAMPLE 16

[0162] N,N′-Bis(4-Phthalimidobutyl)mesitylenesulfonamide (37).

[0163] NaH (60%, 1.6 g, 40 mmol) was added to 35 [Schreine-makers, Recl.Trav. Chim. Pays-Bas Belg., Vol. 16, supra] (2.72 g, 13.5 mmol) in DMF(60 mL) at 0° C. After the mixture was stirred at 0° C. for 30 minutes,N-(4-bromobutyl)phthalimide (11.51 g, 40 mmol) in DMF (20 mL) wasintroduced. The mixture was stirred at room temperature for 1 hour andat 60° C. over-night. Following the workup procedure of 43, columnchromatography (25:1 CHCl₃/acetone) gave 3.77 g (46%) of 37 as a whitepowder: NMR δ 1.51-1.54 (m, 8H), 2.18 (s, 3H), 2.57 (s, 6H), 3.18-3.24(m, 4H), 3.55-3.60 (m, 4H), 6.86 (s, 2 H), 7.69-7.25 (m, 4H), 7.82-7.85(m, 4H). HRMS calcd. for C₃₃H₃₆N₃O₆S 602.2325 (M+H), found 602.2320(M+H).

EXAMPLE 17

[0164] N,N′-Bis(4-aminobutyl)mesitylenesulfonamide (40).

[0165] Hydrazine monohydrate (0.82 g, 16 mmol) was added to a suspensionof 37 (3.5 g, 5.8 mmol) in absolute EtOH (100 mL), and the mixture wasstirred at 65° C. for 24 hours. After cooling, the solid was filteredand washed with EtOH (2×10 mL). The combined filtrate was concentratedand purified by column chromatography (6:1 MeOH/concentrated NH₄OH) toproduce 1.50 g (76%) of 40 as a viscous oil: NMR δ 1.37 (quintet, 4H),1.52 (quintet, 4H), 2.30 (s, 3H), 2.54 (t, 4H, J=7), 2.58 (s, 6H), 3.20(t, 4H, J=7), 7.04 (s, 3H).

EXAMPLE 18

[0166] Homospermidine Trihydrochloride (15).

[0167] HBr (30% in HOAc, 30 mL), 40 (1.50 g, 4.39 mmol) and phenol (4.49g, 48 mmol) in CH₂Cl₂ (20 mL) were reacted, and product was isolated bythe procedure of 2 to afford 0.86 g (73%) of 15 as white crystals: NMR(D₂O) δ 1.73-1.80 (m, 8H), 3.03-3.14 (m, 8H). Anal. (C₈H₂₄Cl₃N₃) C, H,N.

EXAMPLE 19

[0168] N¹,N⁵,N⁹-Tris(mesitylenesulfonyl)homospermidine (32).

[0169] Mesitylenesulfonyl chloride (6.71 g, 30.7 mmol) and 40 (4.76 g,14 mmol) in CH₂Cl₂ (30 mL) and 1 N NaOH (35 mL) were combined and workedup by the method of 31. Column chromatography (4:1 toluene/EtOAc)produced 3.06 g (31%) of 32 as a white foam: NMR δ 1.32-1.38 (m, 4H),1.44-1.54 (m, 4H), 2.28-2.29 (2 s, 9H), 2.54 (s, 6H), 2.60 (s, 12H),2.79 (quartet, 4H), 3.09 (t, 4H, J 7), 4.70-4.80 (br s, 2H), 6.90 (s,2H), 6.92 (s, 4H). Anal. (C₃₅H₅ N₃O₆S₃) C, H, N.

EXAMPLE 20

[0170] N¹,N⁹-Dimethyl-N¹,N⁵,N⁹-tris (mesitylenesulfonyl)homospermidine(50).

[0171] NaH (60%, 0.17 g, 4.2 mmol), 32 (1.28 g, 1.8 mmol), andiodomethane (0.25 mL, 4.0 mmol) in DMF (50 mL) were reacted and workedup as was 43. Column chromatography (2:1 toluene/EtOAc) gave 1.14 g(86%) of 50 as an oil: NMR δ 1.38-1.44 (m, 8H), 2.28 (s, 3H), 2.30 (s,6H), 2.57 (s, 18 H), 2.62 (s, 6H), 3.03-3.14 (m, 8H), 6.93-6.94 (2 s,6H). Anal. (C₃₇H₅₅N₃O₆S₃) C, H, N.

EXAMPLE 21

[0172] N¹,N⁹-Dimethylhomospermidine Trihydrochloride (16).

[0173] HBr (30% in HOAc, 30 mL), 50 (1.12 g, 1.52 mmol), and phenol (5.4g, 57 mmol) in CH₂Cl₂ (25 mL) were reacted, and product was isolated bythe procedure of 2 to provide 354 mg (79%) of 16 as plates: NMR (D₂O) δ1.78 (m, 8H), 2.73 (s, 6H), 3.08-3.12 (m, 8H). Anal. (C₁₀H₂₈Cl₃N₃) C, H,N.

EXAMPLE 22

[0174] N¹,N⁹-Diethyl-N¹,N⁵,N⁹-tris (mesitylenesulfonyl) homospermidine(51).

[0175] NaH (80%, 0.264 g, 8.8 mmol) was added to 35 [Schreinemakers,Recl. Trav. Chim. Pays-Bas Belg., Vol. 16, supra] (0.796 g, 4 mmol) inDMF (60 mL) at 0° C. After the mixture was stirred at 0° C. for 30minutes, 58 [Bergeron et al, J. Med. Chem., Vol. 37, supra]. (3.19 g,8.8 mmol) in DMF (15 mL) was added. The mixture was heated at 75° C.overnight and worked up by the procedure of 43. Column chromatography(3:1 hexane/EtOAc) gave 2.82 g (93%) of 51 as an oil: NMR δ 0.96 (t,6H), 1.20-1.40 (m, 8H), 2.25 (s, 9H), 2.55 (s, 18H), 2.85-3.20 (m, 12H),6.90 (s, 6H). Anal. (C₃₉H₅₉N₃O₆S₃) C, H, N.

EXAMPLE 23

[0176] N¹,N⁹-Diethylhomospermidine Trihydrochloride (17).

[0177] HBr (30% in HOAc, 20 mL), 51 (1.87 g, 2.45 mmol), and phenol (4.4g, 49 mmol) in CH₂Cl₂ (20 mL) were reacted, and product was isolated bythe procedure of 2 to give 493 mg (62%.) of 17 as plates: NMR (D₂O) δ1.30 (s, 6H), 1.55-1.90 (m, 8H), 2.95-3.20 (m, 12H). Anal. (C₁₂H₃₂Cl₃N₃)C, H, N.

EXAMPLE 24

[0178] N¹,N⁹-Dipropyl-N¹,N⁵,N⁹-tris(mesitylenesulfonyl)homospermidine(52).

[0179] NaH (60%, 0.17 g, 4.2 mmol), 32 (1.28 g, 1.8 mmol), and1-iodopropane (0.39 mL, 4.0 mmol) in DMF (50 mL) were reacted and workedup using the procedure of 43. Column chromatography (4:1 hexane/EtOAc)gave 1.26 g (89%) of 52 as an oil: NMR δ 0.74 (t, 6H, J=7), 1.26-1.45(m, 12H), 2.29 (s, 9H), 2.55 (s, 18H), 2.98-3.13 (m, 12H), 6.87 (s, 6H).Anal. (C₄₁H₆₃N₃O₆S₃) C, H, N.

EXAMPLE 25

[0180] N¹,N⁹-Dipropylhomospermidine Trihydrochloride (19).

[0181] HBr (30% in HOAc, 30 mL), 52 (1.24 g, 1.56 mmol), and phenol (5.4g, 57 mmol) in CH₂Cl₂ (25 mL) were reacted, and product was isolated bythe procedure of 2 to give 430 mg (78%) of 19 as plates: NMR (D₂O) δ0.98 (t, 6H, J=7), 1.70 (m, 4H), 1.76-1.80 (m, 8H), 3.02 (t, 4H, J=7),3.08-3.12 (m, 8H). Anal. (C₁₄H₃₆Cl₃N₃) C, H, N.

EXAMPLE 26

[0182] N-(3-Cyanopropyl)mesitylenesulfonamide (36).

[0183] NaH (60%, 2.0 g, 50 mmol), 35 [Schreinemakers, Recl. Trav. Chim.Pays-Bas Belg., Vol. 16, supra] (10.0 g, 50 mmol), and4-bromobutyronitrile (4 mL, 40 mol) in DMF (100 mL) were combined. Themixture was heated at 80° C. overnight and worked up by the procedure of43. Column chromatography (4:3 hexane/EtOAc) gave 5.04 g (38%) of 36 asan oil: NMR δ 1.78 (s, 3H), 2.25 (s, 3H), 2.35 (t, 2H, J=7), 2.95 (q,2H), 5.05 (br t, 1H), 6.90 (s, 2H). Anal. (C₁₃H₁₈N₂O₂S) C, H, N.

EXAMPLE 27

[0184] N—(4-Cyanobutyl)-N—(3-cyanopropyl) mesitylenesulfonamide (38).

[0185] NaH (60%, 0.90 g, 23 mmol), 36 (5.02 g, 18.85 mmol), and5-bromovaleronitrile (2.4 mL, 21 mmol) in DMF were combined and workedup by the method of 43. Column chromatography (1:1 hexane/EtOAc)provided 5.20 g (79%) of 38 as an oil: NMR δ 1.49-1.66 (m, 4H), 1.82 (m,2H), 2.22 (t, 2H, J=7), 2.25 (t, 2H, J=7), 2.29 (s, 3H), 2.57 (s, 6H),3.19 (t, 2H, J=7), 3.29 (t, 2H, J=7), 6.95 (s, 2H). Anal. (C₁₈H₂₅N₃O₂S)C, H, N.

EXAMPLE 28

[0186] 6-(Mesitylenesulfonyl)-1,6,12-Triazadodecane (41).

[0187] Raney nickel (W-2 grade, 7.60 g) and concentrated NH₄OH (10 mL)were successively added to 38 (5.06 g, 14.6 mmol) in CH₃OH (30 mL) andTHF (30 mL) in a 200 mL Parr bottle, and a slow stream of NH₃ wasbubbled through the mixture for 30 minutes at 0° C. After hydrogenationin a Parr bottle was carried out at 50-55 psi for 8 hours, thesuspension was filtered through Celite, and the solvents were removed invacuo to give 4.70 g (91%) of 41 as an oil: NMR δ 1.14-1.24 (m, 10H),2.25 (s, 3 H), 2.6 (s, 6H), 3.05-3.25 (m, 8H), 3.45 (s, 4H), 6.9 (s,2H).

EXAMPLE 29

[0188] 1,6,12-Triazadodecane Trihydrochloride (20).

[0189] HBr (30% in HOAc, 33 mL), 41 (2.43 g, 6.83 mmol), and phenol (6g, 60 mmol) in CH₂Cl₂ were reacted, and product was isolated by theprocedure of 2 to give 0.97 g (50%) of 20 as a hygroscopic solid: NMR(D₂O) δ 1.47 (m, 2H), 1.70-1.80 (m, 8H), 3.00-3.10 (m, 8H). Anal.(C₉H₂₆Cl₃N₃) C, H, N.

EXAMPLE 30

[0190] 1,6,12-Tris(mesitylenesulfonyl)-1,6,12-Triazadodecane (33).

[0191] Mesitylenesulfonyl chloride (4.29 g, 19.6 mmol) and 41 (3.17 g,8.92 mmol) in CH₂Cl₂ (40 mL) and 1 N NaOH (20 mL) were combined andworked up by the method of 31. Column chromatography (4:3 hexane/EtOAc)generated 5.66 g (88%) of 33 as an oil: NMR δ 1.12-1.17 (m, 2H),1.34-1.51 (m, 8H), 2.29 (s, 3H), 2.30 (s, 6H), 2.55 (s, 6H), 2.60-2.62(2s, 12H), 2.77-2.81 (m, 4H), 3.06 (t, 2H, J =7), 3.11 (t, 2H, J=7),4.50-4.60 (m, 2H), 6.92 (s, 2H), 6.95 (s, 2H). Anal. (C₃₆H₅₃N₃O₆S₃) C,H, N.

EXAMPLE 31

[0192] 2,7,13-Tris(mesitylenesulfonyl)-2,7,13-Triazapentadecane (53).

[0193] NaH (60%, 0.28 g, 6.9 mmol), 33 (2.16 g, 3.0 mmol), andiodomethane (6.1 mL, 9.8 mmol) in DMF (30 mL) were combined and workedup by the method of 43. Column chromatography (7:3 hexane/EtOAc) gave1.90 g (85%) of 53 as an oil: NMR δ 1.08-1.16 (m, 2H), 1.38-1.50 (m, 8H), 2.28-2.29 (2s, 9H), 2.57-2.58 (2 s, 18H), 2.63 (s, 3H), 2.65 (s, 3H),3.02-3.14 (m, 8H), 6.95 (s, 6H); HRMS calcd. for C₃₈H₅₈N₃O₆S₃ 748.3487(M+H), found 748.3483 (M+H).

EXAMPLE 32

[0194] 2,7,13-Triazatetradecane Trihydrochloride (21).

[0195] HBr (30% in HOAc, 45 mL), 53 (1.85 g, 2.47 mmol), and phenol (8.5g) in CH₂Cl₂ (20 mL) were reacted, and product was isolated by theprocedure of 2 to give 529 mg (69%) of 21 as crystals: NMR (D₂O) δ1.42-1.52 (m, 2H), 1.69-1.81 (m, 8H), 2.73-2.74 (2s, 6H), 3.03-3.12 (m,8H). Anal. (C₁₁H₃₀Cl₃N₃) C, H, N.

EXAMPLE 33

[0196] 4,9,15-Tris(mesitylenesulfonyl)-4,9,15-Triazaheptadecane (54).

[0197] NaH (60%, 0.273 g, 6.84 mmol), 33 (2.24 g, 3.11 mmol), and1-iodopropane (0.67 mL, 6.9 mmol) in DMF (30 mL) were combined andworked up by the method of 43. Column chromatography (3:1 hexane/EtOAc)provided 2.01 g (80%) of 54 as an oil: NMR δ 0.71-0.78 (m, 6H),1.01-1.11 (m, 2H), 1.34-1.48 (m, 12H), 2.29 (s, 6H), 2.57-2.58 (2 s,18H), 2.98-3.13 (m, 12H), 6.92 (s, 6H). Anal. (C₄₂H₆₅N₃O₆S₃H₂O ) C, H,N.

EXAMPLE 34

[0198] 4,9,15-Triazaoctadecane Trihydrochloride (23).

[0199] HBr (30% in HOAc, 45 mL), 48 (1.99 g, 2.47 mmol), and phenol (8.5g) in CH₂Cl₂ (20 mL) were reacted, and product was isolated by theprocedure of 2 to give 852 mg (83%) of 23 as plates: NMR (D₂O) δ 0.97(s, 6H), 1.40-1.51 (m, 2H), 1.66-1.80 (m, 12 H), 2.98-3.15 (m, 12H).Anal. (Cl₅H₃₈Cl₃N₃) C, H, N.

EXAMPLE 35

[0200] N,N-Bis(4-cyanobutyl)mesitylenesulfonamide (39).

[0201] NaH (80%, 1.22 g, 51 mmol), 35 [Schreinemakers, Recl. Trav. Chim.Pays-Bas Belg., Vol. 16, supra] (5.0 g, 25 mmol), and5-chlorovaleronitrile (6.5 g, 55 mmol) in DMF (50 mL) were combined. Themixture was heated at 60° C. overnight and worked up by the procedure of43. Column chromatography (7:3 hexane/EtOAc) yielded 6.31 g (70%) of 39as an oil: NMR δ 1.57 (m, 4H), 1.66 (m, 4H), 2.26 (t, 4H, J=7), 2.60 (s,6H), 3.22 (t, 4H, J=7), 6.98 (s, 2H). Anal. (C₁₉H₂₇N₃O₂S) C, H, N.

EXAMPLE 36

[0202] 7-Mesitylenesulfonyl-1,7,13-triazatridecane (42).

[0203] Raney nickel (W-2 grade, 2.9 g) and 39 (5.69 g, 15.8 mmol) inconcentrated NH₄OH (10 mL) and CH₃OH (60 mL) were saturated with NH₃ as41. The mixture was shaken with hydrogen at 50-55 psi in a 200 mL Parrbottle for 42 hours. The suspension was filtered through Celite, and thesolvents were removed in vacuo. The residue was passed through a shortsilica gel column (EtOH then 5% concentrated NH₄OH/EtOH) to give 5.71 g(98%) of 42 as a light yellow oil: NMR δ 1.17 (m, 4H), 1.47 (m, 8H),2.27 (s, 3H), 2.57 (s, 6H), 2.61 (m, 4H), 3.13 (t, J=7.5, 4H), 6.91 (s,2H); HRMS calcd. for C₁₉H₃₆N₃O₂S 370.2528 (M+H), found 370.2530 (M+H).

EXAMPLE 37

[0204] 1,7,13-Triazatridecane Trihydrochloride (24).

[0205] HBr (30% in HOAc, 26 mL), 42 (2.0 g, 5.42 mmol), and phenol (4.8g, 51 mmol) in CHCl₃ (40 mL) were reacted, and product was isolated bythe method of 2 to give 0.97 (61%) of 24 as a white solid: NMR (D₂O) δ1.45 (m, 4H), 1.70 (m, 8H), 3.01 (m, 8H). Anal. (C₁₀H₂₈Cl₃N₃) C, H, N.

EXAMPLE 38

[0206] 1,7,13-Tris(mesitylenesulfonyl)-1,7,13-triazatridecane (34).

[0207] Mesitylenesulfonyl chloride (4.52 g, 20.7 mmol) and 42 (3.47 g,9.4 mmol) in CH₂Cl₂ and 1 N NaOH (30 mL) were combined and worked up bythe method of 31. Column chromatography (3:2 hexane/EtOAc) gave 6.44 g(93%) of 34 as a white solid: NMR δ 1.16 (m, 4H), 1.39 (m, 8H), 2.30 (s,3H), 2.31 (s, 6H), 2.57 (s, 6H), 2.62 (s, 12H), 2.81 (d of t, 4 H), 3.10(t, 4H, J=7), 4.49 (br t, 2H), 6.95 (s, 2H), 6.97 (s, 4H); HRMS calcd.for C₃₇H₅₆N₃O₆S₃ 734.3331 (M+H), found 734.3351 (M+H).

EXAMPLE 39

[0208] 2,8,14-Tris(mesitylenesulfonyl)-2,8,14-triazapentadecane (55).

[0209] NaH (80%, 0.207 g, 6.9 mmol), 34 (1.58 g, 2.16 mmol), andiodomethane (0.30 mL, 4.8 mmol) in DMF (30 mL) were reacted and workedup as was 43. Column chromatography (5:2 hexane/EtOAc) gave 1.51 g (92%)of 55 as an oil: NMR δ 1.06-1.18 (m, 4H), 1.40-1.52 (m, 8H), 2.29 (s,9H), 2.59 (s, 18H), 2.66 (s, 6H), 3.03-3.14 (m, 8H), 6.95 (s, 6H). Anal.(C₃₉H₅₉N₃O₆s₃) C, H, N.

EXAMPLE 40

[0210] 2,8,14-Triazapentadecane Trihydrochloride (25).

[0211] HBr (30% in HOAc, 30 mL), 55 (1.48 g, 1.94 mmol), and phenol (5.2g, 55 mmol) in CH₂Cl₂(30 mL) were reacted, and product was isolated bythe method of 2 to produce 480 mg (76%) of 25 as needles: NMR (D₂O) δ1.4-1.5 (quintet, 4H), 1.7-1.8 (quintet, 8H), 2.7 (s, 6H), 3.05 (t, 8H,J=7). Anal. (C₁₂H₃₂Cl₃N₃) C, H, N.

EXAMPLE 41

[0212] 3,9,15-Tris(mesitylenesulfonyl)-3,9,15-triazaheptadecane (56).

[0213] NaH (80%, 0.52 g, 17 mmol), 34 (3.2 g, 4.36 mmol), and iodoethane(1.5 g, 9.6 mmol) in DMF (20 mL) were reacted and worked up by themethod of 43. Column chromatography (4:1 hexane/EtOAc) gave 2.91 g (85%)of 56 as a white solid: mp 60-62° C.; NMR δ 1.01 (t, 6H, J=7), 1.08 (m,4H), 1.42 (m, 8H), 2.29 (s, 9H), 2.57 (s, 6H), 2.58 (s, 12H), 3.07 (t,4H, J=7), 3.11 (t, 4H, J=7), 3.17 (q, 4H), 6.92 (s, 6H). Anal.(C₄₁H₆₃N₃O₆S₃) C, H, N.

EXAMPLE 42

[0214] 3,9,15-Triazaheptadecane Trihydrochloride (26).

[0215] HBr (30% in HOAc, 20 mL), 56 (2.9 g, 3.67 mmol), and phenol (3.25g, 34.5 mmol) in CHCl₃ (27 mL) were reacted, and product was isolated bythe method of 2 to give 1.0 g (77%) of 26 as white crystals: NMR (D₂O) δ1.28 (t, 6H, J=7), 1.45 (m, 4H), 1.73 (m, 8H), 3.08 (m, 12H). Anal.(C₁₄H₃₆Cl₃N₃) C, H, N.

EXAMPLE 43

[0216] 4,10,16-Tris(mesitylenesulfonyl)-4,10,16-triazanonadecane (57).

[0217] NaH (80%, 198 mg, 6.6 mmol), 34 (1.51 g, 2.06 mmol), and1-iodopropane (0.44 mL, 4.5 mmol) in DMF (40 mL) were reacted and workedup by the method of 43. Column chromatography (7:2/hexane/EtOAc)provided 1.61 g (95%) of 57 as an oil: NMR δ 0.75 (t, 6H, J=7),1.02-1.14 (m, 4H), 1.36-1.52 (m, 12H), 2.30 (s, 9H), 2.60 (s, 18H),3.02-3.16 (m, 12H), 6.95 (s, 6H). Anal. (C₄₃H₆₇N₃O₆S₃) C, H, N.

EXAMPLE 44

[0218] 4,10,16-Triazanonadecane Trihydrochloride (27).

[0219] HBr (30% in HOAc, 30 mL), 57 (1.58 g, 1.93 mmol), and phenol (5.2g, 55 mmol) were reacted, and product was isolated by the method of 2 togive 579 mg (79%) of 27 as plates: NMR (D₂O) δ 0.95 (t, 6H, J=7),1.38-1.49 (m, 4H), 1.62-1.77 (m, 12 H), 2.95-3.05 (m, 12H). Anal.(C₁₆H₄₀Cl₃N₃) C, H, N.

EXAMPLE 45

[0220] N-(5-Bromopentyl)-N-ethylmesitylenesulfonamide (61).

[0221] NaH (80%, 1.26 g, 42 mmol), 60 [Schreinemakers, Recl. Trav. Chim.Pays-Bas Belg., Vol. 16, supra] (6.82 g, 30.0 mmol), and1,5-dibromopentane (49 mL, 0.36 mol) in DMF (100 mL) were combined. Themixture was heated at 74° C. overnight and worked up by the procedure of43. Column chromatography (7:1 hexane/EtOAc) produced 7.87 g (70%) of 61as an oil: NMR δ 1.00 (t, 3H, J=7), 1.30-1.75 (m, 6H), 2.20 (s, 3H),2.50 (s, 6H), 3.02-3.30 (m, 6H), 6.80 (s, 2H); HRMS calcd. forC₁₆H₂₇BrNO₂S 376.0946 (M+H), found 376.0960 (M+H).

EXAMPLE 46

[0222] N¹,N⁴-Bis(mesitylenesulfonyl)-N¹-(tert-butoxycarbonyl)-N⁴-ethyl-1,4-diaminobutane (62).

[0223] NaH (80%, 0.45 g, 23 mmol) was added to 59 [Bergeron et al, J.Med. Chem., Vol. 37, supra]. (3.45 g, 11.5 mmol) in DMF (100 mL) at 0°C. After the mixture was stirred at 0° C. for 40 minutes, 58 [Bergeronet al, J. Med. Chem., Vol. 37, supra]. (5.00 g, 13.8 mmol) in DMF (10mL) was added. The mixture was heated at 60° C. for 18 hours and thenworked up by the method of 43. Column chromatography with (20:1toluene/EtOAc) gave 6.44 g (96%) of 62 as an oil: NMR δ 1.12 (t, 3H,J=7), 1.20 (s, 9H), 1.55-1.65 (m, 4H), 2.29 (s, 2H), 2.30 (s, 2H), 2.52(s, 4H), 2.62 (s, 4H), 3.16-3.24 (m, 2H), 3.30 (q, 2H), 3.70 (m, 2H),6.94 (s, 4H).

EXAMPLE 47

[0224] N¹,N⁴-Bis (mesitylenesulfonyl)-N⁴-ethyl-1,4-diaminobutane (63).

[0225] TFA (70 mL) was slowly dripped into a solution of 62 (6.20 g,10.6 mmol) in CH₂Cl₂ (30 mL) at 0° C. After the solution was stirred at0° C. for 20 minutes and at room temperature for 30 minutes, solventswere removed by rotary evaporation. The residue was basified to pH >8with saturated NaHCO₃ and extracted with CH₂Cl₂ (4×100 mL). Removal oforganic extracts led to 5.10 g (100%) of 63 as a foam: NMR δ 0.97 (t,3H, J=7), 1.20-1.50 (m, 4H), 2.25 (s, 6H), 2.50-2.55 (2 s, 12H),2.95-3.25 (m, 6H), 4.45 (t, 1H), 6.85 (s, 4H). Anal. (C₂₄H₃₆N₂O₄S₂) C,H, N.

EXAMPLE 48

[0226] 3,8,14-Tris(mesitylenesulfonyl)-3,8,14-Triazahexadecane (64).

[0227] NaH (80%, 0.41 g, 14 mmol) was added to 63 (5.10 g, 10.6 mmol) inDMF (50 mL) at 0° C. After the mixture was stirred at 0° C. for 30minutes, 61 (4.80 g, 12.7 mmol) in DMF (10 mL) was added. The mixturewas heated at 89° C. overnight and then worked up by the method of 43.Column chromatography (12:1 toluene/EtOAc) gave 5.43 g (66%) of 64 as anoil: NMR δ 0.9-1.1 (m, 6H), 1.2-1.5 (m, 10H), 2.25 (s, 6H), 2.30 (s,3H), 2.55 (s, 18H), 2.9-3.2 (m, 12H), 6.85 (s, 6H). Anal. (C₄₀H₆₁N₃O₆S₃)C, H, N.

EXAMPLE 49

[0228] 3,8,14-Triazahexadecane Trihydrochloride (22).

[0229] HBr (30% in HOAc, 100 mL), 64 (5.4 g, 7.0 mmol), and phenol (25g, 0.28 mol) were reacted, and product was isolated by the method of 2to give 1.63 g (69%) of 22 as plates: NMR (D₂O) δ 1. 38 (t, 3H, J 7),1.39 (t, 3H, J=7), 1.60-1.70 (m, 10H), 3.02-3.15 (m, 12H). Anal.(C₁₃H₃₄Cl₃N₃) C, H, N.

EXAMPLE 50

[0230] N¹-Triphenylmethyl-1,3-diaminopropane (68).

[0231] A solution of triphenylmethyl chloride (6.97 g, 25 mmol) inCH₂Cl₂ (100 mL) was added dropwise to a rapidly stirred solution of1,3-diaminopropane (9.86 g, 133 mmol) in CH₂Cl₂ (100 mL). After stirringat room temperature for 2 days, 1 N NaOH (50 mL) was added to themixture, which was extracted with CHCl₃ (3×50 mL). Organic extracts werewashed with 100 mL of H₂O and brine. After solvent removal, columnchromatography (3% concentrated NH₄OH/MeOH) gave 6.32 g (80%) of 68 as awhite solid: mp 59-61° C. [Parg et al, “A SemiconductingLangmuir-Blodgett Film of a Non-amphiphilic Bis-tetrathiofulvaleneDerivative,” J. Mater. Chem., Vol. 5, pages 1609-1615 (1995); mp 59-61°C.]; NMR δ 1.35-1.65 (m, 6H), 2.18 (t, 2H, J=7), 2.77 (s, 1H), 7.13-7.24(m, 9H), 7.44-7.46 (m, 6H). Anal. (C₂₂H₂₄N₂) C, H, N.

EXAMPLE 51

[0232] N¹-Mesitylenesulfonyl-N³-triphenylmethyl-1,3-diaminopropane (70).

[0233] Mesitylenesulfonyl chloride (5.25 g, 24 mmol) and 68 (6.30 g, 20mmol) in CH₂Cl₂ (30 mL) and 1 N NaOH (27 mL) were combined and worked upby the method of 31. Column chromatography (7:2 hexane/EtOAc) gave 8.37g (84%) of 70 as a white solid: NMR δ 1.56-1.66 (m, 3H), 2.17 (t, 2H,J=7), 2.29 (s, 3H), 2.60 (s, 6H), 3.08 (q, 2H), 5.25 (br t, 1H), 6.93(s, 2H), 7.15-7.28 (m, 9H), 7.35-7.39 (m, 6H). Anal. (C₃₁H₃₄N₂O₂S) C, H,N.

EXAMPLE 52

[0234] N¹-Triphenylmethyl-1,4-diaminobutane (69).

[0235] A solution of triphenylmethyl chloride (59.94 g, 0.215) in CH₂Cl₂(500 mL) was added dropwise to a rapidly stirred solution of1,4-diaminobutane (96.47 g, 1.094 mol) in CH₂Cl₂ (1.1 L) over a periodof 2 hours. The reaction mixture was stirred at room temperature for 3days and was worked up following the method of 68 to give a quantitativeyield of 69 as an oil which was used directly in the next step: NMR δ1.39-1.54 (m, 7H), 2.12 (t, 2H, J=7), 2.62 (t, 2H, J=7), 7.13-7.28 (m, 9H), 7.41-7.47 (m, 6H).

EXAMPLE 53

[0236] N¹-Mesitylenesulfonyl-N⁴-triphenylmethyl-1,4-diaminobutane (71).

[0237] Mesitylenesulfonyl chloride (3.1 g, 14 mmol) and 69 (3.39 g, 10.3mmol) in CH₂Cl₂ (20 mL) and 1 N NaOH (15 mL) were combined and worked upby the method of 31. Column chromatography (1:3 hexane/EtOAc) furnished3.79 g (72%) of 71 as a white solid: NMR δ 1.41-1.50 (m, 5H), 2.03-2.08(t, 2H, J=7), 2.27 (s, 3H), 2.62 (s, 6H), 2.87 (q, 2H), 4.41 (br t, 1H),6.93 (s, 2H), 7.15-7.28 (m, 9H), 7.40-7.44 (m, 6H). Anal. (C₃₂H₃₆N₂O₂S)C, H, N.

EXAMPLE 54

[0238] N-Propylmesitylenesulfonamide (65).

[0239] Mesitylenesulfonyl chloride (12.0 g, 55 mmol) and propylamine(2.96 g, 50 mmol) in CH₂Cl₂ (60 mL) and 1 N NaOH (60 mL) were combinedand worked up by the method of 31. Column chromatography (3:1hexane/EtOAc) afforded 8.44 g (85%) of 65 as a crystalline solid: mp53-54° C.; NMR δ 0.86 (t, 3H, J=7), 1.44-1.51 (m, 2H), 2.30 (s, 3H),2.64 (s, 6H), 2.86 (q, 2H), 4.40 (br t, 1H), 6.96 (s, 2H). Anal.(C₁₂H₁₉NO₂S) C, H, N.

EXAMPLE 55

[0240] N-(3-Bromopropyl)-N-propylmesitylenesulfonamide (66).

[0241] NaH (60%, 0.34 g, 8.4 mmol), 65 (1.7 g, 7.0 mmol), and1,3-dibromopropane (17.0 g, 84 mmol) in DMF (30 mL) were combined andworked up by the method of 43. Column chromatography (6:1 hexane/EtOAc)produced 1.82 g (80%) of 66 as an oil: NMR δ 0.79 (s, 3H), 1.48-1.56 (m,2H), 2.04-2.10 (m, 2H), 2.30 (s, 3H), 2.60 (s, 6H), 3.09-3.14 (m, 2H),3.29-3.36 (m, 4H). Anal. (C₁₅H₂₄BrNO₂S) C, H, N.

EXAMPLE 56

[0242] N-(4-Bromobutyl)-N-propylmesitylenesulfonamide (67).

[0243] NaH (60%, 0.70 g, 17 mmol), 65 (3.5 g, 14.5 mmol), and1,4-dibromobutane (37.6 g, 174 mmol) in DMF (40 mL) were combined andworked up by the method of 43. Excess 1,4-dibromobutane was removed by aKugelrohr apparatus under high vacuum. Column chromatography (7:1hexane/EtOAc) produced 5.21 g (95%) of 67 as an oil: NMR δ 0.79 (t, 3H,J=7), 1.46-1.54 (m, 2H), 1.64-1.78 (m, 4H), 2.30 (s, 3H), 2.60 (s, 6H),3.11 (t, 2H, J=7), 3.21 (t, 2H, J=7), 3.31 (t, 2H, J=7), 6.93 (s, 2H).Anal. (C₁₆H₂₆BrNO₂S) C, H, N.

EXAMPLE 57

[0244]6,10-Bis(mesitylenesulfonyl)-1-triphenylmethyl-1,6,10-triazatridecane(72).

[0245] NaH (60%, 0.24 g, 5.96 mmol) was added to 71 (2.55 g, 4.97 mmol)in DMF (40 mL) at 0° C. After the mixture was stirred at 0° C. for 20minutes, 66 (1.80 g, 4.97 mmol) in DMF (20 mL) was added. The mixturewas stirred at room temperature for 1 day and then worked up followingthe method of 43. Column chromatography (20:1 toluene/EtOAc) produced3.72 g (94%) of 72 as an oil: NMR δ 0.73 (t, 3H, J=7), 1.26-1.70 (m,8H), 2.01 (t, 2H, J=7), 2.26 (s, 3 H), 2.27 (s, 3H), 2.54 (s, 12H),2.96-3.04 (m, 8H), 6.90 (s, 4H), 7.18-7.45 (m, 15H). Anal.(C₄₇H₅₉N₃O₄S₂) C, H, N.

EXAMPLE 58

[0246]5,10-Bis(mesitylenesulfonyl)-1-triphenylmethyl-1,5,10-triazatridecane(73).

[0247] NaH (60%, 0.22 g, 5.42 mmol), 70 (2.25 g, 4.52 mmol) in DMF (40mL), and 67 (1.70 g, 4.52 mmol) in DMF (20 mL) were reacted and workedup following the method of 72. Column chromatography (25:1toluene/EtOAc) produced 2.87 g (80%) of 73 as an oil: NMR δ 0.75 (t, 3H,J=7), 1.41-1.46 (m, 9H), 1.97 (t, 2H, J=7), 2.26 (s, 6H), 2.53 (s, 6H),2.56 (s, 6H), 3.04 (t, 2H, J=7), 3.10-3.15 (m, 6H), 6.88 (s, 2H), 6.90(s, 2H), 7.15-7.38 (m, 15H). Anal. (C₄₇H₅₉N₃O₄S₂) C, H, N.

EXAMPLE 59

[0248]6,11-Bis(mesitylenesulfonyl)-1-triphenylmethyl-1,6,11-triazatetradecane(74).

[0249] NaH (60%, 0.12 g, 2.90 mmol), 71 (1.24 g, 2.42 mmol) in DMF (30mL), and 67 (0.91 g, 2.42 mmol) in DMF (10 mL) were reacted and workedup following the method of 72. Column chromatography (25:1toluene/EtOAc) gave 1.67 g (85%) of 74 as an oil: NMR δ 0.74 (t, 3H,J=7), 1.35-1.45 (m, 11H), 2.00 (t, 2H, J=7), 2.25 (s, 3H), 2.28 (s, 3H),2.55 (s, 6H), 2.56 (s, 6H), 2.98-3.08 (m, 8H), 6.90 (s, 2H), 6.91 (s,2H), 7.15-7.44 (m, 15H). Anal. (C₄₈H₆₁N₃O₄S₂) C, H, N.

EXAMPLE 60

[0250] N¹-Propylspermidine Trihydrochloride (12).

[0251] HBr (30% in HOAc, 45 mL), 72 (3.70 g, 4.66 mmol), and phenol (8.4g, 89 mmol) in CH₂Cl₂ (50 mL) were reacted, and product was isolated bythe method of 2 to give 1.02 g (74%) of 12 as plates: NMR (D₂O) δ 0.98(t, 3H, J=7) 1.66-1.79 (m, 6H), 2.06-2.17 (m, 2H), 3.01-3.19 (m, 10H).Anal. (C₁₀H₂₈Cl₃N₃) C, H, N.

EXAMPLE 61

[0252] N⁸-Propylspermidine Trihydrochloride (13).

[0253] HBr (30% in HOAc, 35 mL), 73 (2.85 g, 3.59 mmol), and phenol (6.5g, 69 mmol) in CH₂Cl₂ (30 mL) were reacted, and product was isolated bythe method of 2 to give 810 mg (76%) of 13 as plates: NMR (D₂O) δ 0.98(t, 3H, J=7), 1.66-1.79 (m, 6H), 2.01-2.12 (m, 2H), 2.99-3.14 (m, 10H).Anal. (C₁₀H₂₈Cl₃N₃) C, H, N.

EXAMPLE 62

[0254] N¹-Propylhomospermidine Trihydrochloride (18).

[0255] HBr (30% in HOAc, 20 mL), 74 (1.65 g, 2.0 mmol), and phenol (3.6g, 38 mmol) in CH₂Cl₂ (20 mL) were reacted, and product was isolated bythe method of 2 to give 268 mg (43%) of 18 as plates: NMR (D₂O) δ 0.98(t, 3H, J=7), 1.66-1.80 (m, 10 H), 2.99-3.11 (m, 10H). HRMS calcd. forC₁₁H₂₈N₃ 202.2283 (free amine, M+H), found 202.2296 (M+H).

EXAMPLE 63

[0256] N¹-Ethylspermidine Trihydrochloride (9).

[0257] Lithium aluminum hydride (1.6 g, 42 mmol) was added toN¹-acetylspermidine dihydrochloride (0.50 g, 1.9 mmol) in THF (300 mL)at 0° C., and the mixture was heated at reflux for 17 hours.

[0258] The reaction was quenched at 0° C. with H₂O (1.6 mL), 15% NaOH(1.6 mL), and H₂O (4.8 mL). Salts were filtered and washed with THF, andsolvent was removed by rotary evaporation. The residue was distilled ina Kugelrohr apparatus under high vacuum (T≦60° C.), and the distillatewas dissolved in EtOH (5 mL) and treated with concentrated HCl (0.5 mL).Recrystallization from aqueous EtOH gave 0.096 g (18%) of 9 as crystals:NMR (D₂O) δ 1.30 (t, 3H, J=7), 1.72-1.83 (m, 4H), 2.05-2.16 (m, 2H),3.02-3.19 (m, 10H). Anal. (C₉H₂₆Cl₃N₃) C, H, N.

EXAMPLE 64

[0259] N⁸-Ethylspermidine Trihydrochloride (10).

[0260] Lithium aluminum hydride (1.73 g, 45.6 mmol) andN⁸-acetylspermidine dihydrochloride (0.54 g, 2.1 mmol) in THF (300 mL)were reacted, and product was isolated by the method of 9 to furnish0.164 g (28%) of 10 as crystals: NMR (D₂O) δ 1.29 (t, 3H, J=7),1.73-1.83 (m, 4H), 2.03-2.16 (m, 2H), 3.05-3.20 (m, 10H). Anal.(C₉H₂₆Cl₃N₃) C, H, N.

EXAMPLE 65

[0261] N¹,N⁴N⁸,N¹¹-Tetrakis(mesitylenesulfonyl)-N¹,N¹¹-dipropylnorspermine (76).

[0262] NaH (60%, 3.60 g, 90.0 mmol), 75 [Bergeron et al, J. Med. Chem.,Vol. 37, supra]. (27.5 g, 30.0 mmol), and 1-iodopropane (7.5 mL, 77mmol) in DMF (200 mL) were combined, and the reaction was worked up bythe method of 48. Column chromatography (5:1 toluene/EtOAc) resulted in27.79 g (92%) of 76 as a white foam: NMR δ 0.72 (t, 6H, J=7), 1.2-1.7(m, 10H), 2.30 (s, 12H), 2.55 (s, 24H), 2.92-3.03 (m, 16H), 6.93 (s,8H). Anal. (C₅₁H₇₆N₄0₈S₄) C, H, N.

EXAMPLE 66

[0263] N¹,N¹¹-Dipropylnorspermine Tetrahydrochloride (28).

[0264] HBr (30% in HOAc, 500 mL), 76 (27.54 g, 27.5 mmol), and phenol(105 g, 1.12 mol) in CH₂Cl₂ (250 mL) were reacted, and product wasisolated by the method of 2 to give 7.82 g (68%) of 28 as white plates:NMR (D₂O) δ 0.98 (t, 6H, J=7), 1.64-1.78 (m, 4H), 2.07-2.21 (m, 6H),3.00-3.25 (m, 16H). Anal. (C₁₅H₄₀Cl₄N₄) C, H, N.

EXAMPLE 67

[0265] N-(5-Chloropentyl)-N-ethylmesitylenesulfonamide (77).

[0266] NaH (80%, 1.34 g, 44.7 mmol) was added to 60 [Bergeron et al, J.Med. Chem., Vol. 37, supra]. (7.68 g, 33.8 mmol) in DMF (130 mL) at 0°C. The mixture was stirred at room temperature for 1 hour, and cooled to0° C. 1,5-Dichloropentane (45 mL, 0.35 mol) was added all at once. Thereaction was stirred at 55° C. for 12 hours and was worked up by themethod of 48. Column chromatography (11.5% EtOAc/hexane) gave 10.54 g(94%) of 77 as an oil: NMR δ 1.07 (t, 3H, J=8), 1.25-1.37 (m, 2H),1.47-1.71 (m, 4H), 2.30 (s, 3H), 2.60 (s, 6H), 3.14-3.28 (m, 4H), 3.44(t, 2H, J=7), 6.94 (s, 2H). Anal. (C₁₆H₂₆ClNO₂S) C, H, N.

EXAMPLE 68

[0267] 3,9,14,20-Tetrakis(mesitylenesulfonyl)-3,9,14,20-tetraazadocosane(79).

[0268] NaH (80%, 1.14 g, 38.0 mmol) was added to 78 [Bergeron et al, J.Med. Chem., Vol. 37, supra]. (5.77 g, 12.7 mmol) in DMF (75 mL) at 0° C.The mixture was stirred at room temperature for 1 hour, and 77 (10.51 g,31.7 mmol) in DMF (55 mL) was added by cannula. The reaction was stirredat 55° C. for 16 hours and was worked up by the method of 48. Columnchromatography (30% EtOAc/hexane) afforded 12.67 g (96%) of 79 as anoil: NMR δ 0.97-1.12 (m, 10H), 1.30-1.47 (m, 12H), 2.29 (s, 12H), 2.56and 2.58 (2 s, 24H), 2.97-3.22 (m, 16H), 6.93 (s, 8H). Anal.(C₅₄H₈₂N₄O₈S₄) C, H, N.

EXAMPLE 69

[0269] 3,9,14,20-Tetraazadocosane Tetrahydrochloride (29).

[0270] HBr (30% in HOAc, 195 mL), 79 (12.66 g, 12.1 mmol), and phenol(33.61 g, 0.357 mol) in CH₂Cl₂ (135 mL) were reacted, and product wasisolated by the method of 2 to provide 4.50 g (81%) of 29 as whitecrystals: NMR (D₂O) δ 1.28 (t, 6H, J=7), 1.40-1.52 (m, 4H), 1.66-1.80(m, 12H), 3.00-3.14 (m, 16H). Anal. (C₁₈H₄₆Cl₄N₄).

ANALYTICAL DATA

[0271] 2 Anal. calcd. for C₈H₂₄Cl₃N₃: C, 35.77;H, 9.00; N, 15.64. Found:C, 35.91;H, 8.96; N, 15.69.

[0272] 4 Anal. calcd. for C₁₀H₂₈Cl₃N₃: C, 40.48;H, 9.51; N, 14.16.Found: C, 40.31;H, 9.36; N, 14.12.

[0273] 5 Anal. calcd. for C₉H₂₆Cl₃N₃: C, 38.24;H, 9.27; N, 14.87. Found:C, 38.15;H, 9.32; N, 14.75.

[0274] 6 Anal. calcd. for C₁₂H₃₂Cl₃N₃: C, 44.38;H, 9.93; N, 12.94.Found: C, 44.42;H, 9.89; N, 12.88.

[0275] 8 Anal. calcd. for C₉H₂₆Cl₃N₃: C, 38.24;H, 9.27; N, 14.86. Found:C, 38.19;H, 9.28; N, 14.79.

[0276] 9 Anal. calcd. for C₉H₂₆Cl₃N₃: C, 38.24;H, 9.27; N, 14.86. Found:C, 38.31;H, 9.23; N, 14.90.

[0277] 10 Anal. calcd. for C₉H₂₆Cl₃N₃: C, 38.24;H, 9.27 N, 14.86. Found:C, 38.28;H, 9.31; N, 14.95.

[0278] 11 Anal. calcd. for C₁₁H₃₀Cl₃N₃: C, 42.52;H, 9.73; N, 13.52.Found: C, 42.59;H, 9.79; N, 13.47.

[0279] 12 Anal. calcd. for C₁₀H₂₈Cl₃N₃: C, 40.48;H, 9.51; N, 14.16.Found C, 40.55;H, 9.45; N, 14.18.

[0280] 13 Anal. calcd. for C₁₀H₂₈Cl₃N₃: C, 40.48;H, 9.51; N, 14.16.Found C, 40.52;H, 9.52; N, 14.09.

[0281] 14 Anal. calcd. for C₁₃H₃₄Cl₃N₃: C, 46.09;H, 10.12; N, 12.40.Found: C, 46.15;H, 10.17; N, 12.43.

[0282] 15 Anal. calcd. for C₈H₂₄Cl₃N₃: C, 35.77;H, 9.00; N, 15.64; S,39.59. Found: C, 35.88;H, 8.91; N, 15.68; S, 39.48.

[0283] 16 Anal. calcd. for C₁₀H₂₈ Cl₃N₃: C, 40.48;H, 9.51; N, 14.16.Found: C, 40.45;H, 9.44; N, 14.09.

[0284] 17 Anal. calcd. for C₁₂H₃₂Cl₃N₃: C, 44.38;H, 9.93; N, 12.94.Found: C, 44.49;H, 9.98; N, 12.96.

[0285] 19 Anal. calcd. for C₁₄H₃₆Cl₃N₃: C, 47.66;H, 10.29; N, 11.91.Found: C, 47.70;H, 10.21; N, 11.86.

[0286] 20 Anal. calcd. for C₉H₂₆Cl₃N₃: C, 38.24;H, 9.27; N, 14.87.Found: C, 38.28;H, 9.22; N, 14.82.

[0287] 21 Anal. calcd. for C₁₁H₃₀Cl₃N₃: C, 42.52;H, 9.73; N, 13.52.Found: C, 42.52;H, 9.69; N, 13.58.

[0288] 22 Anal. calcd. for C₁₃H₃₄Cl₃N₃: C, 46.09;H, 10.12; N, 12.40.Found: C, 46.20;H, 10.08; N, 12.47.

[0289] 23 Anal. calcd. for C₁₅H₃₈Cl₃N₃: C, 49.11;H, 10.44; N, 11.45.Found: C, 49.02;H, 10.40; N, 11.42.

[0290] 24 Anal. calcd. for C₁₀H₂₈Cl₃N₃: C, 40.48;H, 9.51; N, 14.16; Cl,35.85. Found: C, 40.63;H, 9.44; N, 14.16; Cl, 35.70.

[0291] 25 Anal. calcd. for C₁₂H₃₂Cl₃N₃: C, 44.38;H, 9.93; N, 12.94.Found: C, 44.33;H, 9.90; N, 12.89.

[0292] 26 Anal. calcd. for C₁₄H₃₆Cl₃N₃: C, 47.66;H, 10.28; N, 11.91; Cl,30.15. Found: C, 47.69;H, 10.24; N, 11.96; Cl, 30.08.

[0293] 27 Anal. calcd. for C₁₆H₄₀Cl₃N₃: C, 50.46;H, 10.59; N, 11.03.Found: C, 50.49;H, 10.55; N, 11.07.

[0294] 28 Anal. calcd. for C₁₅H₄₀Cl₄N₄: C, 43.07;H, 9.64; N, 13.39.Found: C, 43.24;H, 9.57; N, 13.44.

[0295] 29 Anal. calcd. for C₁₈H₄₆Cl₄N₄: C, 46.96;H, 10.07; N, 12.17.Found: C, 47.08;H, 9.98; N, 12.18.

[0296] 30 Anal. calcd. for C₃₃H₄₇N₃O₆S₃: C, 58.47;H, 6.99; N, 6.20.Found: C, 58.36;H, 6.95; N, 6.18.

[0297] 31 Anal. calcd. for C₄H₄₉N₃O₆S₃: C, 59.02;H, 7.14; N, 6.07.Found: C, 58.74;H, 7.12; N, 5.99.

[0298] 32 Anal. calcd. for C₃₅H₅₁N₃O₆S₃: C, 59.55;H, 7.28; N, 5.95.Found: C, 59.34;H, 7.29; N, 5.92.

[0299] 33 Anal. calcd. for C₃₆H₅₃N₃O₆S₃: C, 60.05;H, 7.42; N, 5.84.Found: C, 59.88;H, 7.41; N, 5.80.

[0300] 36 Anal. calcd. for C₁₃H₁₈N₂O₂S: C, 58.62;H, 6.81; N, 10.52.Found: C, 58.52;H, 6.86; N, 10.46.

[0301] 38 Anal. calcd. for C₁₈H₂₅N₃O₂S: C, 62.22;H, 7.25; N, 12.09; S,8.87. Found: C, 62.24;H, 7.28; N, 11.99.

[0302] 39 Anal. calcd. for C₁₉H₂₇N₃O₂S: C, 63.13;H, 7.53; N, 11.62; S,8.87. Found: C, 63.31;H, 7.68; N, 11.43; S, 8.97.

[0303] 43 Anal. calcd. for C₃₅H₅₁N₃O₆S₃: C, 59.55;H, 7.28; N, 5.95.Found: C, 59.50;H, 7.33; N, 5.88.

[0304] 44 Anal calcd. for C₃₇H₅₅N₃O₆S₃: C, 60.54;H, 7.55; N, 5.72.Found: C, 60.64;H, 7.54; N, 5.73.

[0305] 45 Anal. calcd. for C₃₆H₅₃N₃O₆S₃: C, 60.05;H, 7.42; N, 5.84.Found: C, 59.79;H, 7.32; N, 5.70.

[0306] 46 Anal. calcd. for C₃₉H₅₉N₃O₆S₃: C, 61.55;H, 7.68; N, 5.52.Found: C, 61.52;H, 7.79; N, 5.55.

[0307] 48 Anal. calcd. for C₃₈H₅₇N₃O₆S₃: C, 61.01;H, 7.68; N, 5.62.Found: C, 61.22;H, 7.76; N, 5.56.

[0308] 49 Anal. calcd. for C₄₀H₆₁N₃O₆S₃: C, 61.90;H, 7.92; N, 5.41.Found: C, 61.71;H, 7.86; N, 5.35.

[0309] 50 Anal. calcd. for C₃₇H₅₅N₃O₆S₃: C, 60.54;H, 7.55; N, 5.72.Found: C, 60.26;H, 7.61; N, 5.63.

[0310] 51 Anal. calcd. for C₃₉H₅₉N₃O₆S₃: C, 61.47;H, 7.80; N, 5.51.Found: C, 61.27;H, 7.89; N, 5.44.

[0311] 52 Anal. calcd. for C₄₁H₆₃N₃O₆S₃: C, 62.32;H, 8.04; N, 5.32.Found: C, 62.19;H, 8.00; N, 5.33.

[0312] 54 Anal. calcd. for C₄₂H₆₅N₃O₆S₃.H₂O: C, 61.35;H, 8.21; N, 5.11.Found: C, 61.34;H, 8.07; N, 5.05.

[0313] 55 Anal. calcd. for C₃₉H₅₉N₃O₆S₃: C, 61.47;H, 7.80; N, 5.51.Found: C, 61.54;H, 7.79; N, 5.51.

[0314] 56 Anal. calcd. for C₄₁H₆₃N₃O₆S₃: C, 62.32;H, 8.04; N, 5.32; S,12.17. Found: C, 62.40;H, 8.08; N, 5.25; S, 12.07.

[0315] 57 Anal. calcd. for C₄₃H₆₇N₃O₆S₃: C, 63.12;H, 8.25; N, 5.14.Found: C, 63.21;H, 8.23; N, 5.04.

[0316] 63 Anal. calcd. for C₂₄H₃₆N₂O₄S₂: C, 59.97;H, 7.55; N, 5.83.Found: C, 59.83;H, 7.56; N, 5.76.

[0317] 64 Anal. calcd. for C₄₀H₆₁N₃O₆S₃: C, 61.90;H, 7.92; N, 5.41.Found: C, 62.03;H, 7.97; N, 5.33.

[0318] 65 Anal. calcd. for C₁₂H₁₉NO₂S: C, 59.72;H, 7.93; N, 5.80. Found:C, 59.69;H, 7.88; N, 5.80.

[0319] 66 Anal. calcd. for C₁₅H₂₄BrNO2S: C, 49.72;H, 6.68; N, 3.87.Found C, 49.97;H, 6.76; N, 3.83.

[0320] 67 Anal. calcd. for C₁₆H₂₆BrNO₂S: C, 51.06;H, 6.96; N, 3.72.Found: C, 51.17;H, 6.95; N, 3.74.

[0321] 68 Anal. calcd. for C₂₂H₂₄N₂: C, 83.50;H, 7.64; N, 8.85. Found C,83.42;H, 7.67; N, 8.86.

[0322] 70 Anal. calcd. for C₃₁H₃₄N₂O₂S: C, 74.67;H, 6.87; N, 5.62.Found: C, 74.62;H, 6.89; N, 5.54.

[0323] 71 Anal. calcd. for C₃₂H₃₆N₂O₂S: C, 74.97;H, 7.08; N, 5.46.Found: C, 74.71;H, 7.12; N, 5.51.

[0324] 72 Anal. calcd. for C₄₇H₅₉N₃O₄S₂: C, 71.09;H, 7.49; N, 5.29.Found C, 71.35;H, 7.53; N, 5.18.

[0325] 73 Anal. calcd. for C₄₇H₅₉N₃O₄S₂: C, 71.09;H, 7.49; N, 5.29.Found C, 71.16;H, 7.46; N, 5.33.

[0326] 74 Anal. calcd. for C₄₈H₆₁N₃O₄S₂: C, 71.34;H, 7.61; N, 5.20.Found C, 71.22;H, 7.64; N, 5.10.

[0327] 76 Anal. calcd. for C₅₁H₇₆N₄O₈S₄: C, 61.17;H, 7.65; N, 5.59.Found: C, 61.21;H, 7.67; N, 5.58.

[0328] 77 Anal. calcd. for C₁₆H₂₆ClNO₂S: C, 57.90;H, 7.90; N, 4.22.Found: C, 57.98;H, 7.82; N, 4.27.

[0329] 79 Anal. calcd. for C₅₄H₈₂N₄O₈S₄: C, 62.16;H, 7.92; N, 5.37.Found: C, 62.30;H, 7.86; N, 5.37.

I claim:
 1. A polyamine which does not occur in nature having theformula:

or a salt thereof with a pharmaceutically acceptable acid wherein: R₁-R₅may be the same or different and are alkyl, aryl, aryl alkyl, cycloalkylor hydrogen; at least one of said R₁ and R₂ and at least one of said R₄and R₅ are not hydrogen, and any of said alkyl chains may optionally beinterrupted by at least one etheric oxygen atom, excludingN¹,N³-diethylspermidine and N¹,N³-dipropylspermidine; and A and B may bethe same or different and are bridging groups including unsubstitutedheterocyclic bridging groups which effectively maintain the distancebetween the nitrogen atoms such that the polyamine: (i) is capable ofuptake by a target cell upon administration of the polyamine to a humanor non-human animal; and (ii) upon uptake by said target cell,competitively binds via an electrostatic interaction between thepositively charged nitrogen atoms to substantially the same biologicalcounter-anions as the intracellular natural polyamines in the targetcell, provided that where A or B is a heterocyclic bridging group, thebridging group is an unsubstituted heterocyclic group incorporating saidN¹, N² or N³ atoms in the heterocyclic ring as an unsubstituted N atom;said polyamine, upon binding to the biological counter-anion in thecell, functions in a manner biologically different than saidintracellular polyamines.
 2. A polyamine according to claim 1, uponbinding to said biological counter-anion in said cell, exerting ananti-neoplastic function.
 3. A polyamine according to claim 1, whereinsaid bridging groups A and B may be the same or different and arealkylene, branched alkylene, cycloalkylene or arylalkylene.
 4. Thepolyamine according to claim 1 having the formula:R₁—N¹H—(CH₂)₃—N²H—(CH₂)₃—N³H—R₂ wherein: R₁ and R₂ may be the same ordifferent and are alkyl having, at most, 10 carbon atoms.
 5. Thepolyamine of claim 4 wherein R₁=R₂=methyl.
 6. The polyamine of claim 4wherein R₁=R₂=ethyl.
 7. The polyamine of claim 4 wherein R₁=R₂=n-propyl.8. The polyamine of claim 4 wherein R₁=H and R₂=ethyl.
 9. The polyamineof claim 4 wherein R₁=H and R₂=n-propyl.
 10. The polyamine according toclaim 1 having the formula: R₁—N¹H—(CH₂)₃—N²H—(CH₂)₄—N³H—R₂ wherein: R₁and R₂ may be the same or different and are alkyl having, at most, 10carbon atoms.
 11. The polyamine of claim 10 wherein R₁=R₂=methyl. 12.The polyamine of claim 10 wherein R₁=R₂=ethyl.
 13. The polyamine ofclaim 10 wherein R₁=R₂=n-propyl.
 14. The polyamine of claim 10 whereinR₁=ethyl and R₂=H.
 15. The polyamine of claim 10 wherein R₁=H andR₂=ethyl.
 16. The polyamine of claim 10 wherein R₁=n-propyl and R₂=H.17. The polyamine of claim 10 wherein R₁=H and R₂=n-propyl.
 18. Thepolyamine according to claim 1 having the formula: R₁—N¹H—(CH₂)₄—N²H—(CH₂)₄—N³H—R₂ wherein: R₁ and R₂may be the same or different and are alkylhaving, at most, 10 carbon atoms.
 19. The polyamine of claim 18 whereinR₁=R₂=methyl.
 20. The polyamine of claim 18 wherein R₁=R2=ethyl.
 21. Thepolyamine of claim 18 wherein R₁=R₂=n-propyl.
 22. The polyamine of claim18 wherein R₁=H and R₂=ethyl.
 23. The polyamine of claim 18 wherein R₁=Hand R₂=n-propyl.
 24. The polyamine according to claim 1 having theformula: R₁—N¹H—(CH₂)₄—N²H—(CH₂)₅—N³H—R₂ wherein: R₁ and R₂ may be thesame or different and are alkyl having, at most, 10 carbon atoms. 25.The polyamine of claim 24 wherein R₁=R₂=methyl.
 26. The polyamine ofclaim 24 wherein R₁=R₂=ethyl.
 27. The polyamine of claim 24 whereinR₁=R₂=n-propyl.
 28. The polyamine according to claim 1 having theformula: R₁—N¹H—(CH₂)₅—N²H—(CH₂)₅—N³H—R₂ wherein: R₁ and R₂ may be thesame or different and are alkyl having at most, 10 carbon atoms.
 29. Thepolyamine of claim 28 wherein R₁=R₂=methyl.
 30. The polyamine of claim28 wherein R₁=R₂=ethyl.
 31. The polyamine of claim 28 whereinR₁=R₂=n-propyl.
 32. A pharmaceutical composition in unit dosage formcomprising a pharmaceutically acceptable carrier and a pharmaceuticallyeffective amount of a polyamine of claim 1 or a salt thereof with apharmaceutically acceptable acid.
 33. The pharmaceutical composition ofclaim 32 comprising an amount of said polyamine or salt pharmaceuticallyeffective to treat a human or non-human patient afflicted with tumorcells sensitive to said polyamine or salt thereof.
 34. A method oftreating a human or non-human patient in need thereof comprisingadministering thereto a pharmaceutically effective amount of a polyamineof claim 1 or a salt thereof with a pharmaceutically acceptable acid.35. The method according to claim 34 comprising administering to saidpatient afflicted with tumor cells sensitive to said polyamine or saltthereof an amount of said polyamine or salt thereof pharmaceuticallyeffective to inhibit the growth of said tumor cells.